Ensuring that neuronal projections form appropriate connections with their intended target is an essential feature of brain development. Neuronal growth cones often travel great distances and encounter numerous decision points where they integrate an array of attractive and repulsive guidance cues that steer axons towards the proper route. Repellent guidance cues that divert axons away from undesirable destinations can be broadly divided into soluble/diffusible factors, which establish an inhibitory concentration gradient, and contact-mediated cues, which normally cause the growth cone to collapse and recover its forward growth in an alternate direction. Since the discovery of the first repulsive guidance cue nearly 20 years ago (Luo et al., 1993
), the effect of repellent cues on growth cone behavior and axon guidance have been studied intensively in many different model systems, most notably in the developing visual pathways.
After exiting the eye, RGC axons from both retinae travel through the optic nerve and converge at the ventral diencephalon to form the optic chiasm. The decision to project ipsilaterally or contralaterally at the optic chiasm is the first step in establishing the binocular pathway and is essential for stereopsis. In mice, which have a relatively small binocular field, the vast majority of RGC axons traverse the chiasm midline with only a small percentage of RGCs projecting ipsilaterally (~3–5%). This ipsilateral projection arises from the peripheral ventrotemporal (VT) retina.
Differential growth cone behavior associated with this chiasm-crossing decision was first examined in DiI-labeled RGC growth cones as they encounter radial glia processes at the optic chiasm midline (Godement et al., 1990
; Guillery et al., 1995
; Marcus et al., 1995
; Mason and Sretavan, 1997
). As RGC growth cones encounter the optic chiasm, their normally smooth extension and streamlined morphology within the optic nerve transforms into cycles of saltatory extension, pausing and retraction, accompanied by adoption of complex morphologies (Godement et al., 1990
; Godement et al., 1994
; Marcus et al., 1995
; Mason and Wang, 1997
; Mason and Erskine, 2000
). Growth cones of VT RGCs usually pause longer than their contralaterally-projecting counterparts and eventually project into the ipsilateral optic tract by consolidation of a backwards-oriented filopodium. These observations led to the idea that VT RGCs are prevented from crossing the chiasm by contact-mediated cues expressed on radial glia cells.
Work in both Xenopus
and mice (Nakagawa et al., 2000
; Williams et al., 2003
) identified members of the EphB family of receptor tyrosine kinases and their ligands, the ephrin-Bs, as playing critical roles in formation of the ipsilateral retinal pathway. During formation of the ipsilateral projection (E13–E16.5 in mice), EphB1 expression is restricted to RGC axons originating from the peripheral VT crescent, whereas ephrin-B2 is expressed by radial glia at the optic chiasm. EphB1−/−
mice have a strongly reduced ipsilateral projection (Williams et al., 2003
), and ectopic expression of EphB1 into non-VT RGCs can reroute these fibers into the ipsilateral optic tract (Petros et al., 2009
However, this apparently simple mechanism is complicated by the fact that RGCs express other EphB receptors that have similar binding affinities for ephrin-B2 (Flanagan and Vanderhaeghen, 1998
): EphB2 is expressed in a high-ventral to low-dorsal gradient in RGCs (Williams et al., 2003
; McLaughlin and O'Leary, 2005
) and EphB3 appears to be expressed homogenously throughout the retina at E14.5 (Birgbauer et al., 2000
; Williams et al., 2003
). Therefore RGC’s exhibit distinct EphB expression profiles dependent on their location within the retina: VT RGCs are EphB1+/EphB2+/EphB3+; ventronasal (VN) RGCs are EphB1−/EphB2+/EphB3+; and dorsotemporal (DT) RGCs are EphB1−/EphB2−/EphB3+. Although combinatorial EphB receptor signaling has not been ruled out, the mild repulsion of VN axons by ephrin-B2 substrates in vitro
(Williams et al., 2003
) and weak rerouting effect of ectopic EphB2 expression in vivo
(Petros et al., 2009
) suggests that EphB1-ephrin-B2 interaction is the primary mechanism for directing the ipsilateral retinal projection. This is in contrast to the retinotectal (McLaughlin and O'Leary, 2005
) and thalamocortical (Vanderhaeghen et al., 2000
; Cang et al., 2005
) projections where gradients of multiple Eph receptors and ephrins direct growth cones to their appropriate destination, making the optic chiasm a relatively simple system in which to study the mechanisms underlying ephrin-induced growth cone behavior.
In this study, we have attempted to broaden our understanding of this mechanism by characterizing the initial effect of ephrin-B2 on growth cone collapse and axon retraction of RGCs from different retinal regions. To this end, we combined time-lapse imaging with the established in vitro
collapse assay (Raper and Kapfhammer, 1990
; Kapfhammer et al., 2007
), allowing one to study the real-time effects of a compound on growth cone behavior. We found that ephrin-B2 induces rapid growth cone collapse and sustained axon retraction of VT RGCs, whereas ephrin-B2 has little effect on DT RGCs. VN RGCs behave similar to VT RGCs at high ephrin-B2 concentrations but are less sensitive than VT RGCs at more moderate ephrin-B2 concentrations. Additionally, we demonstrate that blockade of Rho-kinase (ROCK), a likely downstream component of the EphB1 signaling cascade, abrogates ephrin-B2-induced axon retraction of VT RGCs but not growth cone collapse. In summary, these findings characterize the differential ephrin-B2-induced responses of RGC growth cones arising from distinct retinal regions, and represent an assay for examining additional downstream signaling components of EphB receptors and their role in RGC axon guidance.