PlexinD1 is expressed by growing axons in two major descending pathways
To visualize potential neuronal targets of Sema3E activity, we analyzed the binding of Sema3E-alkaline phosphatase (Sema3E-AP) fusion protein to sections of mouse brain at E17.5, when major descending nerve tracts have begun to form (Figs. , ). Sema3E-AP delineated two axonal pathways. A first pathway — through the internal capsule (ic; ) and the cerebral peduncle (cp; ) — marks a trajectory common to corticofugal and striatonigral projections (). A second Sema3E-AP-delineated pathway - through the fimbria (fim; ), the fornix (f; ) and the post-commissural fornix (pf; ) — marks the trajectory of the subiculo-mammillary tract ().
In the vascular system, the principal functional receptor for Sema3E is PlexinD1 (Gu et al., 2005
). No Sema3E-AP binding could be detected using sections of PlexinD1-/-
embryonic brain (Suppl. Fig. 1U, V
), demonstrating that PlexinD1 is also the predominant binding partner for Sema3E in the brain. PlexinD1
mRNA was detected in the ventrolateral regions of the cortex (including the piriform, perirhinal and insular cortices), in the striatum and in the pyramidal layer of the subiculum (, ; Suppl. Fig. 2
) during the formation of the descending pathways in the forebrain (at E15.5-E17.5). Moreover, PlexinD1 protein was detected along the length of axons in the cerebral peduncle and post-commissural fornix (, ; Suppl. Fig. 3
). Anterograde DiI tracing from the ventrolateral cortex, striatum and hippocampal formation at E17.5 labeled the same axon tracts as did Sema3E-AP binding (Suppl. Fig. 1
) and PlexinD1 antibody (, ), confirming the identity of the pathways identified.
We therefore focused our analysis on the role of Sema3E in controlling the growth and trajectory of axons along these two descending forebrain pathways.
Correlation between Npn-1 expression and differing growth responses to Sema3E in vivo
To understand how Sema3E influences the growth of these forebrain projections, we first identified potential sources of Sema3E in proximity to growing axons in each pathway (E15.5-P2). At each stage examined, the highest levels of Sema3E
expression were detected in the globus pallidus and the thalamic reticular nucleus, identified by their expression of Er81
homeogenes, respectively (; Suppl. Fig. 2A-C
; Jones and Rubenstein, 1994; Sussel et al., 1999
). These nuclei surround the internal capsule, through which corticofugal and striatonigral fibers pass on their way to the cerebral peduncle, and thus could repel and channel axons at this decision point (). Along the subiculo-mammillary pathway, Sema3E
expression was detected only in the pyramidal cell layers in the CA1 and CA3 fields of the hippocampus, adjacent to the subiculum (; Suppl. Fig. 2M-O
; Chedotal et al., 1998
; Miyazaki et al., 1999b
). Efferent axons from CA3 and CA1 regions could act as sources of Sema3E, since they project along the same initial pathway as subiculo-mammillary axons (). No other sites of Sema3E
expression were observed along the pathway of hippocampal projections, at any stage examined.
Thus, axons in both sets of descending pathways pass close to cells that express Sema3E. Corticofugal and striatonigral axons avoid potential sources of Sema3E by growing between them (see scheme in ) whereas subiculo-mammillary axons grow alongside the axons of neurons that express Sema3E (see scheme in ). A possible explanation for the different growth behaviors of these classes of neurons in vivo
is that neurons in each pathway express different receptor complexes for Sema3E, involving receptor components in addition to PlexinD1. Neuropilin-1 (Npn-1) is required for Sema3E activity in some neurons (Miyazaki et al., 1999b
) and so we monitored its expression in each neuronal population (E15.5-P2). In the cortex, no Npn-1
was detected in PlexinD1
-expressing ventrolateral regions of the cortex; and expression of Npn-1
was restricted to more dorsal areas of the cortex (; Suppl. Fig. 2G-I
was also absent from the striatum (; Suppl. Fig. 2G-I
). Furthermore, Npn-1 protein was not detected on PlexinD1-positive axons of either the corticofugal or striatonigral tracts (; Suppl. Fig. 3
), although it was clearly expressed in unidentified adjacent tracts in the pons (). In contrast, Npn-1
was observed in the subiculum, overlapping with the domain of PlexinD1
expression (; Suppl. Fig. 2S-U
), and Npn-1 protein was expressed along the full length of the subiculo-mammillary tract (; Suppl. Fig. 3
). Moreover, PlexinD1 and Npn-1 were co-expressed by individual subicular axon fascicles within the postcommissural fornix ().
Together, these findings reveal that forebrain neurons exhibit two contrasting patterns of semaphorin ligand-receptor expression. A sub-population of corticofugal axons and striatonigral axons express PlexinD1 but not Npn-1 and evade sources of Sema3E surrounding the internal capsule. In contrast, subiculo-mammillary axons express both PlexinD1 and Npn-1, and grow together with axons of CA1 and CA3 neurons that express Sema3E (schemes in Figs. , ).
Differential axonal responses to loss of Sema3E in vivo
We next examined the role of Sema3E and PlexinD1 in the development of descending forebrain axonal projections by comparing axon trajectories in wild-type, Sema3E-/-
mice. The gross morphology of the internal capsule labeled with Sema3E-AP was very similar in E17.5 wild-type and Sema3E-/-
embryos (). However, in all mutant embryos, ectopic axon fascicles labeled by Sema3E-AP projected through the thalamic reticular nucleus () and terminated abnormally in the dorsal midbrain (; scheme in ; Suppl. Fig. 9
; pattern observed in 5/5 Sema3E-/-
and 0/7 wild-type littermate embryos). In the absence of specific markers, it was not possible to distinguish whether the ectopic fibers reflected misguidance of corticofugal or striatonigral axons, or both.
Opposite effects of Sema3E inactivation on descending pathways correlate with presence or absence of Npn-1
Along the subiculo-mammillary pathway of Sema3E-/-
embryos, no Sema3E-AP binding was detected in the region of the postcommissural fornix at E17.5 (; pattern observed in 5/5 Sema3E-/-
and 0/7 wild-type littermate embryos), although the number of subicular neurons was unchanged (Suppl. Fig. 4
). This observation suggests that subicular axons fail to project along the subiculo-mammillary tract in the absence of Sema3E. To assess this possibility, we performed anterograde DiI tracing from the dorsal hippocampus at E17.5. In Sema3E-/-
mutants the postcommissural fornix contained few, if any, labeled axons, whereas it was robustly labeled in wild-type controls (; phenotype observed in 6/6 mutant and 0/4 wild-type embryos). As a control to determine whether the phenotype in Sema3E-/-
embryos was specific to subicular axons, we examined the precommissural fornix and hippocampal commissure, through which CA1 and CA3 efferents project (), and found that labeling was normal (data not shown). The absence of labeled axons in the postcommissural fornix raised the possibility that subicular neurons instead innervated ectopic targets. Along the proximal subiculo-mammillary tract it was not possible to distinguish these fibers from CA1 projections, which are also potentially labeled by DiI injection. However, no ectopic DiI-labeled axons were detected distal to the precommissural fornix in mutant embryos.
To examine whether Sema3E acts through PlexinD1 to control axonal growth, we examined PlexinD1-/- embryos at E17.5. Strikingly, anterograde DiI tracing of fibers projecting from the dorsal hippocampus revealed that, as in PlexinD1-/- mutants, very few axons reached the postcommissural fornix (; phenotype observed in 3/3 PlexinD1-/-, 0/1 wild-type and 0/2 heterozygote embryos). In the absence of specific markers, other than PlexinD1, for cortical and striatal efferents that may be affected in PlexinD1-/- mutants, the characterization of these pathways in the mutant embryos is not currently feasible.
Thus, in the absence of Sema3E signaling, some corticofugal and/or striatonigral axons, which are normally routed through the internal capsule, grow instead into the thalamic reticular nucleus and dorsal midbrain, regions from which they are normally excluded (scheme in ). In contrast, subiculo-mammillary axons in Sema3E/PlexinD1 mutant embryos showed reduced growth towards the postcommissural fornix (scheme in ). One explanation for these findings would be that Sema3E is normally repulsive for corticofugal and/or striatonigral axons but attractive and/or growth-promoting for subiculo-mammillary axons, a possibility we test below.
Adult Sema3E null mice show behavioral defects reflecting decreased anxiety and memory impairment
We also asked whether the perturbations observed in Sema3E-/- embryos persist at later stages. Sections of internal capsule from wild-type and mutant pups at P4 were incubated with Sema3E-AP (; Table 1). The exuberant projections of axons into the thalamic reticular nucleus of Sema3E-/-embryos could no longer be detected at P4, suggesting that the defect in corticofugal and/or striatonigral projections had been corrected by elimination of the ectopic axons. In contrast, neurofilament immunostaining on coronal brain sections of P30 Sema3E-/- mice revealed a subiculo-mammillary tract that, as in mutant embryos, was abnormally thin (; observed in 3/3 mutant and 0/3 wild-type animals). The persistence of this axonal project defect provided an opportunity to determine whether Sema3E inactivation affected adult behavior.
Adult Sema3E null-mutant mice show behavior consistent with mammillary body denervation
Experimental lesions of the mammillary bodies lead to defects in emotional behavior and working memory (Beracochea and Krazem, 1991
; Santin et al., 1999
). To detect potential emotional alterations in Sema3E-/-
mice, anxiety was evaluated by testing the mice in an elevated plus maze, consisting of two enclosed and two open arms. When placed in the maze for 8 minutes, Sema3E-/-
mice visited the open arms () and their extremities () >3-fold more frequently than did wild-type controls. They also spent >5-fold longer periods in the open arms (). All effects of genotype were highly significant (n=11 mice per genotype; p<0.016 by ANOVA). Increased exploration did not reflect a general increase in motility since spontaneous activity of mutant mice in isolation was not altered (data not shown). Sema3E-/-
mice therefore show behavior suggestive of reduced anxiety levels.
The performance of Sema3E-/- mice was also evaluated in the Morris water maze, which provides a measure of spatial memory. Mutant mice showed normal performance when the platform in the tank was visible, demonstrating that Sema3E-/- mice have no deficit in swimming ability, motivation, or other performance parameters (data not shown). The mutant mice were also normal in the acquisition and retrieval of spatial reference memory tested in the hidden platform version of the task ().
We therefore used a variant of the Morris water maze in which the position of the platform is changed from day to day (Malleret et al., 1999
). Each day, initial placement onto the daily platform (sample stage) was followed by four trials of escape latency. In this paradigm, mice have to solve a new spatial problem each day, while extinguishing their long-term memory of the previously learned platform location. Results for each successive trial were averaged across 5 successive days (). Wild-type mice learned from their initial placement and performed correctly in the first daily trial. In contrast, Sema3E-/-
mice exhibited 30% longer latency than wild-type mice on the first daily trial (p=0.03). Nevertheless, they were able to improve their trial-to-trial performance on a given day, showing a correct short-term processing of spatial information in subsequent trials (). Thus the null-mutant mice showed intact spatial reference memory but a moderately impaired spatial working memory. The reduced growth of subiculo-mammillary axons in Sema3E-/-
embryos therefore leads to an adult behavioral pattern consistent with dysfunction of the mammillary bodies
Both positive and negative effects of Sema3E on axonal growth require PlexinD1
We next addressed the mechanisms underlying Sema3E signaling in the different forebrain neuronal populations. To determine directly how co-expression of Npn-1 with PlexinD1 affects growth responses to Sema3E, we cultured neurons from the ventrolateral region of the cortex expressing PlexinD1
but not Npn-1,
or from the subiculum expressing PlexinD1
(). The hippocampus proper was taken as a control, since it expresses undetectable levels of PlexinD1
(). We first determined whether the profile of receptor expression in these cultures faithfully reflected the in vivo
pattern. As expected from the high density of PlexinD1
expression in the subdissected regions in vivo
(insets in Figs. , ), PlexinD1 protein was expressed by 90% of cultured neurons from the ventrolateral cortex and 92% of subicular neurons, whereas Npn-1 could only be detected in subicular cultures, in which it was expressed by 90% of neurons (Suppl. Fig. 5
PlexinD1 is required for both positive and negative effects of Sema3E
To measure the effects of Sema3E on axonal elongation, dissociated neurons were cultured in the presence or absence of recombinant Sema3E (5 nM) and axon length was quantified after 2-3 days. The number of living neurons was not affected by Sema3E (data not shown), but in the presence of Sema3E, cortical axon length was reduced by 45% (p<0.001; ). In contrast, the mean length of subicular axons was increased by 55% in the presence of Sema3E (p<0.001; ). The length of hippocampal axons was unaffected by the addition of Sema3E ().
To determine whether the opposite effects of Sema3E on axonal length of cortical and subicular axons are also reflected in its effects on axonal guidance, explants of ventrolateral cortex, subiculum or hippocampus from PlexinD1-/- or control embryos were co-cultured with aggregates of HEK293T cells expressing Sema3E or control cells (). After 2 days in vitro, axons from cortical explants co-cultured with control HEK293T cells extended in a radially symmetric manner. When confronted with Sema3E-expressing cells, axonal length in the quadrant closest to the explant was reduced by 40% compared to that in the opposite quadrant (p<0.05; ). Thus, Sema3E repels the axons of cortical neurons. In contrast, subicular axons were attracted to Sema3E. Axonal length was ~70% greater in the quadrant proximal to Sema3E-secreting cells as compared to the distal quadrant (p<0.001; ).
We next asked whether PlexinD1 was required for both repulsive and attractive effects of Sema3E on axons. Explants of either ventrolateral cortex or subiculum taken from PlexinD1-/- mice showed radial growth despite the presence of Sema3E-expressing cells (), demonstrating that PlexinD1 is required for both the repulsive and attractive effect. Neurons from each region therefore use PlexinD1 to signal contrasted axonal responses to Sema3E - repulsion/growth inhibition for ventrolateral cortex, and attraction/growth stimulation for subiculum - consistent with the in vivo phenotypes of Sema3E-/- and PlexinD1-/- embryos.
To examine whether the altered behavior of neurons isolated from PlexinD1-/-
mice directly reflected the absence of PlexinD1 signaling, rather than an earlier developmental defect, we studied the effects of knock-down of PlexinD1 in dissociated neurons, using two different siRNAs that lowered PlexinD1 expression in COS-7 cells by >95%, without affecting Npn-1 levels (Suppl. Fig. 6A-C
). The siRNAs were co-electroporated with a GFP reporter vector, and only cells that had been transduced (as indicated by GFP fluorescence) and had a clear neurite >1 cell diameter in length were included in the analysis. As with PlexinD1-/-
neurons, knockdown of PlexinD1 to low levels abrogated both positive and negative effects of Sema3E on axonal length (). As a control, we grew cultures of neurons isolated from the dorsal part of the cortex, which exhibits minimal expression of PlexinD1
in mouse embryos (see Suppl. Fig. 2E
). The same siRNAs had no effects on inhibition of axon growth by Sema3A, indicating that effects of PlexinD1 knockdown were specific to Sema3E (Suppl. Fig. 7
; Bagnard et al., 1998
These findings indicate that cortical and subicular neurons show intrinsic differences in their PlexinD1-dependent responses to Sema3E in vitro. Sema3E acts as a repulsive/inhibitory signal for cortical axons but as an attractive/promoting cue for subicular axons. Since both axonal populations express PlexinD1, but only subiculo-mammillary processes express Npn-1, our data suggest a model in which “gating” by Npn-1 of the response of PlexinD1 to Sema3E transforms the repulsive signal mediated by PlexinD1 into an attractive effect.
Npn-1 is required for stimulatory, but not inhibitory, effects of Sema3E on axonal growth
To analyze the role of Npn-1 in each forebrain neuronal population, we used neutralizing antibodies to the extracellular domain of Npn-1. These antibodies completely block the inhibitory response of hippocampal axons to Sema3A, which is known to act through Npn-1 (; Chedotal et al., 1998
). Npn-1 blocking antibodies had no effect on the inhibitory growth response to Sema3E of cortical axons. In contrast, the antibodies completely inhibited the axonal growth-promoting effects of Sema3E on subicular neurons (). Since it has been suggested that neuropilin-2 may also form a complex with PlexinD1 (Gitler et al., 2004
), antibodies to Npn-2 were used as a control. As expected, Npn-2 antibodies completely blocked the response of hippocampal neurons to Sema3F (; Bielenberg et al., 2004
). However, they did not interfere with the response of subicular neurons to Sema3E (), despite the fact that Npn-2 is expressed by this population (data not shown). These results suggest that Npn-1, but not Npn-2, is a necessary component of the Sema3E signaling mechanism involved in increased axonal growth, but not growth inhibition.
Levels of Npn-1 gate the responses of cortical and subicular axons to Sema3E
To exclude the possibility of indirect steric hindrance by Npn-1 antibodies, we modulated Npn-1 levels in cultured neurons from ventrolateral cortex and subiculum using RNA interference. Two different siRNA sequences reduced Npn-1 levels to <5% of normal values (Suppl. Fig. 6D-F
). Strikingly, in dissociated cultures from E17.5 subiculum, knockdown of Npn-1 transformed the positive growth response to Sema3E into an inhibitory one (p<0.001; ). The same siRNAs had no effect on the inhibition of cortical axonal growth by Sema3E (). Thus, Npn-1 is necessary for the growth-promoting effects of Sema3E on subicular neurons.
To determine whether Npn-1 is alone sufficient to confer on cortical axons a positive growth response to Sema3E, we electroporated cortical neurons with an expression vector encoding Npn-1, co-transfected with a GFP reporter vector. Cortical neurons over-expressing Npn-1 showed an 80% increase in axonal length when cultured in the presence of Sema3E, whereas neurons transfected with the GFP vector alone showed a 40% decrease in length in response to Sema3E (p<0.001; ). This Npn-1-dependent growth-promoting effect required PlexinD1 function, since it could be inhibited using siRNAs specific to PlexinD1 (). Thus both Npn-1 and PlexinD1 are required to initiate a growth-promoting response to Sema3E. Indeed, Npn-1 could be co-precipitated with PlexinD1 after heterologous expression of epitope-tagged proteins in COS-7 cells (), supporting the idea that Npn-1 and PlexinD1 form part of a receptor complex (Gitler et al., 2004
). In contrast, although it could be co-immunoprecipitated with PlexinD1 in the COS-7 system (Suppl. Fig. 10
), Npn-2 did not convert the growth inhibitory response of cortical neurons to Sema3E into growth promotion (not shown).
The extracellular domains of Npn-1 and PlexinD1 interact to signal growth
Mechanism of gating of PlexinD1 signaling by Npn-1
Npn1-PlexinD1 interactions have previously been shown to require the Sema domain in the extracellular portion of PlexinD1 (Gitler et al., 2004
). We therefore asked whether interactions between the extracellular domains of PlexinD1 and Npn-1 were sufficient to convert axonal responses to Sema3E from inhibition to growth enhancement. We used Npn1-Fc - the Npn-1 extracellular domain fused to a human Fc immunoglobulin domain - which functions as an agonist, since it rescues the vascular phenotype of Npn1-/-
embryos in vivo
(Yamada et al., 2001
). As in earlier experiments, addition of Sema3E to control cortical neurons led to a 40% decrease in mean axonal length (). In contrast, addition of Npn1-Fc to the culture medium of cortical neurons led to a 60% increase in mean axon length in response to Sema3E (p<0.001; ), just as observed following forced expression of full-length Npn-1 (). A strikingly similar switch in growth responses to Sema3E from inhibition to stimulation was observed following addition of Npn1-Fc to striatal neurons (p<0.01; ), which express PlexinD1 but not Npn-1 (). Moreover, in co-culture experiments using cortical explants, Npn1-Fc caused growing axons to be attracted towards a source of Sema3E (p<0.05; ). Thus, exposure of neurons to the extracellular domain of Npn-1 is sufficient to switch their response to PlexinD1 signaling.
We investigated the possibility that Npn-1 might affect Sema3E signaling by modulating levels of PlexinD1. However, co-expression of Npn-1 in COS-7 cells did not alter surface levels of PlexinD1 (Suppl. Fig. 8B
). Moreover, the Kd
of Sema3E binding to PlexinD1 is unchanged in the presence of full length Npn-1, whereas the number of binding sites is modified (Suppl. Fig. 8C
). This result showed that changes in Kd
are not necessary for switching to occur but left open the possibility that the gating might involve changes in Bmax
. However, addition of Npn1-Fc to COS-7 cells expressing PlexinD1 induced no significant change in the number of binding sites for Sema3E (Suppl. Fig. 8E
). Thus the switch in PlexinD1 signaling triggered by Npn-1 likely reflects changes in intracellular signaling events, which are currently unknown.
Together, our findings demonstrate that Npn-1 is necessary and sufficient to “gate” the normally inhibitory/repulsive signal generated by binding of Sema3E to PlexinD1. Strikingly, the extracellular domain of Npn-1 is sufficient to determine whether PlexinD1 signals repulsion or attraction (). The expression of Npn-1 in subicular neurons but not in cortical or striatal neurons therefore appears to explain the differential response of these neuronal populations to Sema3E in vitro, and their contrasting responses to Sema3E inactivation in vivo.