Reverse signaling through ephrin-As on RGC axons is implicated in the development of the retinocollicular map (Rashid et al., 2005
) and in several other axonal projections (Knoll et al., 2001
; Cutforth et al., 2003
; Marquardt et al., 2005
). However, because ephrin-As are GPI-linked proteins and lack an intracellular domain, they require a transmembrane signaling partner to initiate the intracellular pathways that carry out their functions. Here we show that p75NTR
is a signaling partner for ephrin-As and activates an intracellular cascade that mediates the repellent effects of ephrin-A reverse signaling on RGC axons required for their proper guidance and mapping.
We show that p75 is expressed in RGCs and that p75 protein is present in their axons in vivo at the appropriate developmental stages to mediate guidance and mapping. In addition, p75 co-localizes with ephrin-As along retinal axons and complexes with ephrin-As in caveolae. Further, this association of p75 and ephrin-A results in a functional signaling complex that when activated by EphA binding to ephrin-As leads to increased levels within caveolae of phosphorylated Fyn. Our demonstration that EphA binds ephrin-A but not p75 indicates that EphAs are not ligands per se for p75, but through its association with ephrin-As, p75 acts as co-receptor, or signaling partner, for them and is required to activate their reverse signaling pathway. We also find that the increased phosphorylation and recruitment of Fyn to caveolae is dependent upon p75, which itself is recruited to caveolae upon EphA binding ephrin-A.
Functional evidence for the p75-ephrin-A signaling complex that we describe is provided by our in vitro axon guidance assays showing that p75 is required for the repulsion of retinal axons by EphA. In contrast, p75 is not required for the repulsion of retinal axons by ephrin-A. These data indicate that p75 is selectively required for the repellent guidance activity mediated by ephrin-A reverse signaling, but is not required for the repellent guidance activity mediated by EphA forward signaling. A recent report suggested that the sensitivity of axons in peripheral nerves to the repellent activity of Sema3A is enhanced in p75 knockout mice (Ben-Zvi et al., 2007
). However, we do not find evidence for an analogous role for p75 in RGC axons; that is, the lack of p75 does not lead to increased sensitivity to ephrin-A in stripe assays and that EphA forward signaling appears unaffected.
We also demonstrate that p75 is required for appropriate topographic mapping of RGC axons in the SC, by analyzing mice constitutively null for p75 or in which floxed alleles of p75 are selectively deleted from retina. In both p75 mutants, essentially all RGC axons aberrantly terminate anterior to their topographically appropriate position in the SC. This anterior shift in the terminations of p75 deficient RGC axons is the predicted outcome if the repellent activity of ephrin-A reverse signaling along the AP axis of the SC is diminished and p75 mediates this signaling (Supplemental Figure 1
). In conclusion, the findings from each set of experiments in our study support the conclusion that p75 complexes with ephrin-A in RGC axon membranes, and that p75 is required for the transduction of a repellent signal to RGC axons when axonally expressed ephrin-A binds EphA.
p75 −/− mice have normal retinal morphology and numbers of RGCs (Harada et al., 2006
) and we find no obvious defects in the retina or SC in either p75 −/− mice or p75 fl/fl; α-cre mice, including the expression of ephrin-As, EphAs, and RGC markers. The α-cre mice used to delete p75 from retina in the conditional p75 knockout mice have the important feature that cre-recombinase is not expressed in the SC or anywhere in the visual pathway outside of the retina (Marquardt et al., 2001
; Baumer et al., 2002
). In addition, the early phases of map development appear similar in p75 mutants compared to wild type mice. These observations, the similarity in mapping defects in the two distinct p75 mutant lines, and the consistency of the mapping defects in p75 mutant mice with results from our in vitro axon guidance assays, show that the aberrant phenotypes are due to the lack of p75 in RGC axons, and are not due to secondary effects.
The aberrant mapping that we observe in the p75 mutant mice is very consistent and is the predicted phenotype for a diminished action of ephrin-A reverse signaling. Our finding that the mapping defect is characterized by the formation of a relatively normal appearing TZ at an aberrant anterior position in the SC indicates that p75 deficient RGC axons are affected in a uniform manner, with essentially all RGC axons exhibiting a diminished response to ephrin-A repulsion. However, the magnitude of the anterior shift of the terminations of p75 deficient RGC axons might be viewed as subtle. It is possible that the anterior shift in terminations in p75 mutant mice is limited by the action of another signaling partner for ephrin-A partially redundant with p75, for example TROY, a transmembrane receptor that shares features with p75 (Park et al., 2005
). But, even if p75 is essentially completely responsible for mediating the repellent effect of ephrin-A reverse signaling, as indicated by our findings using the protein stripe assay, we would expect the observed phenotype because other mechanisms that influence AP mapping are still intact, including competitive interactions between axons, EphA forward signaling, and “redundant” mapping mechanisms suggested to explain phenotypes in ephrin-A and EphA mutants (Frisen et al., 1998
; Feldheim et al., 2000
; Pfeiffenberger et al., 2006
). Because essentially all p75 deficient RGC axons are affected in a uniform manner, the competitive balance between them would be retained, and would act to limit the magnitude of the anterior shift of their terminations. For example, competitive interactions limit the aberrant posterior shift in the terminations of RGC axons genetically engineered to express higher levels of EphA, and therefore experience higher levels of repellent EphA forward signaling (Brown et al., 2000
The first experimental evidence for ephrin-A reverse signaling in the retinocollicular projection was reported by Rashid et al. (2005)
based on the protein stripe assay and an analysis of EphA7 knockout mice. EphA7, as well as EphA3 and EphA4, is expressed in a high to low AP gradient in the SC, but EphA7 is not expressed in retina. In EphA7 knockout mice, a proportion of nasal RGC axons target to anterior SC rather than their appropriate TZ in posterior SC (Rashid et al. 2005
). However, the proportion of nasal RGC axons that mis-target is small, and most project to their topographically correct site in posterior SC. In contrast, in p75 mutants we find that essentially all RGC axons mis-target anterior to their appropriate position in the SC, but the magnitude of the anterior shift is relatively small. Thus, in terms of appearance of the ectopic terminations, the EphA7 phenotype stands out because the subset of aberrantly projecting axons substantially mis-target, and the ectopic TZ that they form can be compared directly to the appropriate TZ that most RGCs form in the same SC. However, in terms of the proportion of axons that exhibit the aberrant phenotype, the p75 phenotype is more robust than the EphA7 phenotype.
Further, the EphA7 mutant phenotype (Rashid et al., 2005
) is intriguing and perhaps unexpected because only one of several EphAs with a high to low AP graded expression is eliminated from the SC, and the small proportion of RGC axons affected mis-target to anterior SC that retains high EphA expression. An interpretation that explains the EphA7 mutant phenotype is that a small subset of RGCs responds differently to EphA7 than to EphA3 and EphA4. Although a basis for such a differential response has not been reported, RGCs are a heterogeneous population, with some RGC subsets comprising as little as 1% of the population (Hattar et al., 2002
The classic function of p75 is as an NTR. BDNF, a neurotrophin ligand for the high affinity NTR, TrkB, also binds p75, and has a general role as a growth promoter of RGC axon arbors in the retinotectal projection in Xenopus (Cohen-Cory and Fraser, 1995
; Alsina et al., 2001
). Thus p75 may have a role as a NTR in retinocollicular development, although the TrkB mutant has been reported to have a normal retinocollicular map (Rohrer et al., 2001
). Regardless, the internal consistency of our findings indicate that the phenotypes we find are due to p75 complexing with ephrin-A to mediate the repellent effect of ephrin-A reverse signaling, rather than p75 mediating growth promoting effects of BDNF. For example, in the stripe assay wild type retinal axons are strongly repelled by EphAs, but p75 null retinal axons are not affected, though in vitro, explants from wild type and p75 null mice extend the same number of axons and their average distance of extension is the same. Further, in vivo, p75 deficient RGC axons exhibit their normal, initially exuberant growth across the SC. Thus, in the absence of p75, axon repulsion due to ephrin-A reverse signaling is lost, but general features of axon growth are normal. In conclusion, p75 deficient retinal axons exhibit normal outgrowth but selectively lose their repellent response to EphAs indicating that our findings are not due to the loss of a trophic effect of p75 but is due to the specific loss of the repellent effect of ephrin-A reverse signaling mediated by p75.
The reports of roles for ephrin-A reverse signaling in the pathfinding of spinal motor axons (Marquardt et al., 2005
) and the mapping of olfactory receptor axons (Cutforth et al., 2003
), which also express p75 (Carson et al., 2005
; Domeniconi et al., 2007
), suggest the possibility that p75 acts as a signaling partner with ephrin-As in other developing projection systems. In addition, Fyn, which is a prominent component of the p75-ephrin-A signaling pathway, is also an important contributor to the signaling pathways of other guidance molecules, including netrin/DCC and sema3A (Sasaki et al., 2002
; Liu et al., 2004
; Meriane et al., 2004
). Thus, it is likely that p75 acts broadly as a partner for ephrin-A reverse signaling and potentially other families of axon guidance molecules and their signaling pathways suggesting that both p75 and Fyn are involved in integrating multiple signaling pathways to provide coherent guidance information.