Waltzing Mice Add a New Twist to the Function of EphB2
Authors: Esther T. Stoeckli
Journal: Neuron (2000)

The Eph receptor tyrosine kinases and their ligands, the ephrins, initially caught the attention of neurobiologists with the discovery that they are involved in the formation of topographic projections in the retinotectal system (recently reviewed by 2Flanagan J.G. Vanderhaeghen P Annu. Rev. Neurosci. 1998; 21: 309-345Crossref PubMed Scopus (942) Google Scholar, 5Holder N. Klein R Development. 1999; 126: 2033-2044Crossref PubMed Google Scholar, 9O'Leary D.D.M. Wilkinson D.G Curr. Opin. Neurobiol. 1999; 9: 65-73Crossref PubMed Scopus (292) Google Scholar). Sperry, in his chemoaffinity hypothesis, proposed that gradients of matching labels in the projecting and target fields allowed axons to project to topographically correct positions. After decades of searching, the Eph receptors (EphRs) and their ligands, the ephrins, were identified as the potential candidates for the molecules proposed by Sperry's chemoaffinity hypothesis, and indeed both in vitro and in vivo studies in the chick and mouse visual systems support a role for these families in topographic map formation. Branching off from these initial studies in the retinotectal system, the ephrins have now been shown to act as contact repellents for a wide variety of axons and cell types. While topographic mapping in the visual system is still the most frequently used model system to study the role of the Eph receptor tyrosine kinases and their ligands, recent work in other systems has implicated this family in a spectrum of other functions, including the guidance and fasciculation of axons, boundary formation in the nervous system, cell migration, and vascular development (3Frisén J. Holmberg J. Barbacid M EMBO J. 1999; 18: 5159-5165Crossref PubMed Scopus (123) Google Scholar, 5Holder N. Klein R Development. 1999; 126: 2033-2044Crossref PubMed Google Scholar). In this issue of Neuron, Cowan and colleagues add to this list a role for EphB2 in guidance of inner ear efferents (IEE) at the midline and in the regulation of ionic homeostasis in the vestibular system of the inner ear. The analysis of midline crossing in the brain and the spinal cord has provided evidence for the model that axon behavior at the midline is regulated by a balance between positive and negative guidance cues (11Stoeckli E.T. Landmesser L.T Curr. Opin. Neurobiol. 1998; 8: 73-79Crossref PubMed Scopus (116) Google Scholar). Genetic, biochemical, and anatomical experiments in both vertebrates and invertebrates have implicated both positive, attractant and negative, repellent molecules in regulating axon pathfinding at the midline. Recently, several papers have pointed to a potential role for Eph/ephrin signaling in regulating guidance at the midline as well. A role for Eph/ephrins at the midline was first proposed in the forebrain, where analysis of a protein null mutation in EphB2 (Nuk) revealed that EphB2 activity is required for the proper formation of the anterior commissure (4Henkemeyer M. Orioli D. Henderson J.T. Saxton T.M. Roder J. Pawson T. Klein R Cell. 1996; 86: 35-46Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Interestingly, while axons in EphB2 mutant mice fail to form a proper anterior commissure, they do not themselves express the EphB2 receptor. Rather, the receptor is expressed in the surrounding tissues of the ventral forebrain and forms a boundary that these axons, which express the B-ephrin “ligands,” avoid. As an added surprise, pathfinding was not affected in mice carrying a mutation that produced a membrane-bound version of EphB2 receptor lacking the cytoplasmic tyrosine kinase domain, suggesting that in the case of the projections of the anterior commissure, the ephrinB “ligands” are the acting receptors that transduce a “reverse” signal from the midline to the axons. Consistent with a role for ephrins in midline guidance, Nakagawa and colleagues showed recently that Eph/ephrin signaling may also be required to set up the pattern of crossed and uncrossed projections at the Xenopus optic chiasm (8Nakagawa S. Brennan C. Johnson K.G. Shewan D. Harris W.A. Holt C.E Neuron. 2000; 25: 599-610Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). At the optic chiasm, axons from specific regions of the retina either cross, projecting to the contralateral side of the brain, or continue projecting ipsilaterally. Nakagawa and colleagues showed that ephrin-B is expressed at the chiasm, and EphB receptors are expressed by retinal axons. Furthermore, misexpression of ephrin-B is sufficient to cause EphB-expressing axons to project ipsilaterally. In this issue, Cowan and colleagues report that the EphBs also guide the pathfinding of yet another population of midline-crossing axons: the IEEs, a population of axons that modulate sensory input to the inner ear. Interestingly, the role of ephrins/EphRs in guiding these crossing axons to the ear represents yet another twist on how ephrins/EphB work to steer axons at the midline. IEEs that have their cell bodies located near the floor plate in rhombomere 4 are a heterogeneous population of axons, as some of them project to the ipsilateral ear, whereas others cross the midline to project to the contralateral ear. In mice lacking the EphB2 receptor (i.e., EphB2 single and EphB2/EphB3 double knockouts), the IEEs were delayed in arrival in the ear, and some of the axons projecting to the contralateral ear were misrouted. Rather than crossing the midline, they extended caudally within the floor plate. Thus, whereas ephrin-B at the Xenopus chiasm may be required to prevent subpopulations of normally ipsilaterally projecting retinal ganglion cell axons from crossing the chiasm inappropriately, in the case of the IEE projections the normally crossed IEEs require EphB expression to direct them across the midline. At first glance, these results in the chiasm and at the floor plate would seem to be contradictory. How could this apparent discrepancy and the IEE phenotype be explained? Do ephrins, as these results might suggest, play a dual role, preventing some axons from crossing while guiding others across? Clearly, neither a repulsive activity nor an attractive mechanism, as suggested for EphBs and B-ephrins in some situations (see 9O'Leary D.D.M. Wilkinson D.G Curr. Opin. Neurobiol. 1999; 9: 65-73Crossref PubMed Scopus (292) Google Scholar, for a discussion and references), can fully explain the IEE phenotype. Similar to the axonal phenotype that was observed at the Drosophila midline in Slit mutants (7Kidd T. Bland K.S. Goodman C.S Cell. 1999; 96: 785-794Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar), in the absence of EphB2 IEE axons grow toward the floor plate but then fail to leave. This phenotype could be explained by a shift in the balance between positive and negative cues derived from the midline, such that in the absence of the repulsive EphB signal, the floor plate is now perceived as less repulsive and more attractive, causing axons to linger at the midline. Since EphB2 is also expressed in the region of the IEE cell bodies, the absence of EphB2, and thus a reduction in surrounding repulsive signals, may even explain why ipsilaterally projecting IEE axons are delayed in arriving in the ear. Explaining the specific signaling mechanisms that underlie the IEE midline phenotype is further complicated by the fact that IEE axons, in addition to expressing EphB2 and presumably EphB3, express EphB1, EphB6, and EphA4 receptors as well as ephrin-B1 ligand and that several Eph receptors (EphB1, EphB2, EphA4) and all B-ephrins are also expressed at the midline. Thus, a multiplicity of interactions between Eph and ephrin family members are possible. In addition, both “forward” and, as in the case of the anterior commissure, “reverse” signaling between receptors and ligands may be involved. The coexpression of B-class ephrins and EphB “receptors” in the same cell may modulate that cell's sensitivity to EphB such that in the absence of EphB receptor, the sensitivity of these axons to midline Eph/ephrin cues is changed. Such ligand modulation of receptor responsiveness has been proposed in the retinotectal system (6Hornberger M.R. Dütting D. Ciossek T. Yamada T. Handwerker C. Lang S. Weth F. Huf J. Wessel R. Logan C. et al.Neuron. 1999; 22: 731-742Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Likewise, ephrin cluster size, i.e., dimeric versus multimeric interactions (10Stein E. Lane A.A. Cerretti D.P. Schoecklmann H.O. Schroff A.D. Van Etten R.L. Daniel T.O Genes Dev. 1998; 12: 667-678Crossref PubMed Scopus (367) Google Scholar), have also been proposed to play a role in regulating ephrin signaling. Thus, the absence of EphB2 in the floor plate or in the axons could result in a change in the distribution of B-ephrins, which in turn changes their availability as ligands for the remaining EphBs as well as for EphA4, the only A-class receptor with a binding affinity for B-ephrins. Clearly, a more detailed analysis of expression patterns, biochemical links, and cell–cell interactions will be required to resolve the precise mechanisms underlying the aberrant behavior of the IEEs. The initial impetus for examining the inner ear of these EphB knockout animals was a strain-specific circling phenotype, which suggested a vestibular, inner ear defect. Since the aberrant pathfinding of IEEs was a transient phenotype that was no longer found in old embryonic and postnatal mice, it seemed unlikely that the axonal defects alone could explain the circling behavior. Further analysis of the inner ear of these mice revealed a severe reduction in the cross-sectional diameter of the semicircular canals and an even more dramatic decrease in the volume of endolymph fluid. This reduction in endolymph fluid was suggested to result in inadequate stimulation of the inner hair cells, and thus to cause the waltzing phenotype of these mice. A link between EphB2 and the function of the vestibular system was suggested by the expression of EphB2 in dark cells lining the endolymph-filled semicircular canals and the observation that these cells had accumulated fluid-filled vesicles in mutant mice. Since dark cells play a role in endolymph production by regulating the establishment and maintenance of its unique ionic composition, it was tempting to speculate that EphB2 may play a role in endolymph production via molecular interactions with channel and anion exchanger proteins prevalent in the dark cells. The PDZ domain–containing proteins Pick-1 and Syntenin, which were known to bind the C termini of EphBs and B-ephrins, became attractive candidates for linking Eph/ephrins with proteins involved in ion homeostasis. A screen for anion exchanger and channel proteins capable of interacting with the PDZ domains of Pick-1 and Syntenin confirmed this hypothesis and identified three aquaporins (AQP1, 2, and 9) that could bind to Pick-1 and the anion exchanger AE2 that could bind to both Pick-1 and Syntenin. Additional biochemical data, as well as colocalization studies, indicate that EphB2 can indeed form a complex with aquaporin-1 and that the two proteins do not interact directly with each other but rather bind to the PDZ domain of homo-oligomerized Pick-1. While the study by Cowan et al. adds a potential role in the regulation of fluid homeostasis to the growing list of functions of EphB receptors, the mechanisms underlying this phenotype remain unclear. A number of issues remain to be addressed. In particular, what is the precise function of EphBs/ephrins in ion exchange? Are they involved in the localization of the aquaporins and ion exchangers on the dark cells? Or do they play an active regulatory role in the function of these channels and transporters? What, if any, is the link between the function of Eph/ephrins in the vestibular system and in axon guidance? In contrast to what has been observed at the anterior commissure, proper guidance of IEE projections at the midline requires the cytoplasmic domain of EphB2. Thus, it is tempting to speculate that interactions between Eph/ephrins and PDZ domain proteins may also play a role in the signaling interactions that govern the complex pathfinding behavior of IEEs at the midline. Given the complexity of interactions between Eph receptors, ephrin ligands, and associating proteins, it seems likely that we will see even more “twists” in this tale in the near future.