Selective expression of plexinA1 by proprioceptive sensory neurons
To identify receptors involved in establishing the trajectory of proprioceptive axons we examined 107 genes encoding putative transmembrane proteins for their profile of expression in e15.5 mouse DRG (Table S1). Of this set, 27 were expressed by subsets of DRG neurons (Table S1). These were analyzed further for expression in the DRG of trkC
mutant mice, where proprioceptive sensory neurons are depleted (Klein et al., 1994
), and in the DRG of ngn1
mutant mice, where proprioceptive sensory neurons are enriched (Ma et al., 1999
). The expression of one gene, plexinA1
, was almost completely eliminated from the DRG of trkC
mutants (; data not shown), and was enriched in the DRG of ngn1
mutants (; data not shown).
To determine whether plexinA1
is expressed by all proprioceptive sensory neurons, we compared the profile of plexinA1
expression with that of parvalbumin
), a marker of all proprioceptive sensory neurons (Honda et al., 1995
; Arber et al., 2000
). We found virtually complete coincidence in expression of plexinA1
in e15.5 DRG neurons (), indicating that plexinA1
is expressed by all proprioceptive neurons. During development, the expression of plexinA1
by DRG neurons was first detected at e11.5, and by e13.5 expression was restricted to ~20% of DRG neurons, a profile that was maintained until at least p8 (; data not shown). Diffuse plexinA1
expression was also detected throughout the gray matter of embryonic spinal cord (; data not shown). The selectivity of plexinA1
expression prompted us to examine the patterns of expression of the eight other mouse plexin
genes. PlexinA2, plexinA3
, and plexinD1
were each expressed by subsets of Pv
-labeled proprioceptive neurons (data not shown), but were also expressed by other classes of DRG neurons (data not shown).
Sema6C and sema6D binding to plexinA1
We also examined the pattern of expression of plexinA1 protein by DRG neurons. Little or no expression of plexinA1 was detected in sensory neuron somata, or in sensory axons in the dorsal roots, between e13.5 and p1 (data not shown). In contrast, plexinA1 was detected on the shafts of TrkC+ proprioceptive axons within the dorsal funiculus, on rostrocaudally oriented axons branches within the dorsal columns (), and on axon collaterals in the dorsal spinal cord (). Thus, the axons of proprioceptive neurons express plexinA1 protein during the initial phase of their projection in the spinal cord. PlexinA1 expression was also detected on the axons of spinal neurons that project in the lateral funiculi and dorsal columns, and by cells in the intermediate and ventral spinal cord (; data not shown).
Sema6C and sema6D are ligands for plexinA1
To define ligands for plexinA1 we examined the binding of class 3 (sema3A, sema3C, sema3E and sema3F) and class 6 (sema6A, sema6B, sema6C, and sema6D) semas to plexins and npns (; data not shown). COS-7 cells transfected with plexinA1, plexinA2, plexinD1, npn1, or npn2 plasmids, were exposed to the ectodomains (ecto) of semas conjugated to alkaline phosphatase (AP) (; data not shown). Binding of AP-sema6Cecto and AP-sema6Decto was detected to plexinA1, but not to plexinA2, plexinD1, npn1, or npn2-transfected COS-7 cells (; data not shown). In contrast, AP-sema6Aecto, AP-sema6Becto, and AP-sema3 proteins did not bind to plexinA1-transfected COS-7 cells (; data not shown). Thus, sema6C and sema6D interact selectively with plexinA1. The interaction of sema6D with plexinA1 has been reported (Toyufuku et al., 2004).
To determine if plexinA1 serves as a receptor for sema6C and sema6D in the developing spinal cord, we examined the binding of AP-sema6Cecto and AP-sema6Decto to sections of spinal cord obtained from wild-type and plexinA1 mutant mice (see below). We detected binding of AP-sema6Cecto and AP-sema6Decto to the dorsal and lateral funiculi of the spinal cord in wild-type mice at e13.5 (), a pattern that matched sites of high level plexinA1 protein expression (). In contrast, there was no detectable binding of AP-sema6Cecto, and a marked reduction in binding of AP-sema6Decto, to sections of spinal cord obtained from plexinA1 mutant mice (). AP-sema3A, AP-sema3F, AP-sema6Aecto and AP-sema6Becto bound in a similar pattern to spinal cord sections obtained from wild-type and plexinA1 mutant mice (; data not shown). These data indicate that plexinA1 is a major target of sema6C and sema6D in the developing spinal cord.
Domains of sema6C/6D expression complement proprioceptive axon tracts
Proprioceptive sensory axons enter the spinal cord at the dorsal root entry zone (drez) and the parental axon shaft gives rise to branches that extend rostrally and caudally in the dorsal funiculus, avoiding the superficial dorsal horn (). With time, the rostrocaudally-oriented shafts and branches of proprioceptive axons shift progressively medially and reach the dorsal column. During their medial translocation, the branches of proprioceptive axons send off collaterals that circumvent the superficial dorsal horn as they project to their intermediate and ventral target zones () (Brown, 1981
). Throughout this developmental program, the shafts, branches and collaterals of proprioceptive axons are excluded from the superficial dorsal horn, the target region of high-threshold cutaneous afferent fibers.
To assess the role of plexinA1 signaling in the patterning of proprioceptive afferent projections, we compared the profiles of expression of sema6C
with the known trajectory of proprioceptive axons. We assessed proprioceptive axon trajectory by monitoring the pattern of GFP immunoreactivity in TrkC::eGFP
BAC transgenic mice (Gong et al., 2003
). At e11.5 to e12.5, sema6C
was expressed by cells in the dorsal rim of the spinal cord (), whereas sema6D
was expressed in a much broader dorsal domain (). At this stage, proprioceptive sensory axons have reached the drez (Ozaki and Snider, 1997
), and are confronted by a dorsal domain of high level sema6C/6D
expression (). By e13.5, the level of sema6C
expression had decreased markedly (), and sema6D
expression had cleared from a medial strip within the dorsal spinal cord (). Thus, the composite patterns of sema6C
at e13.5 reveal the emergence of a sema6C/D
-sparse zone that coincides with the position of entry of proprioceptive axon collaterals (). By e14.5, sema6C
expression was barely detectable (), and high level sema6D
expression was restricted to an extreme dorsal rim that is flanked by the shafts of proprioceptive axons (). Together, these observations reveal a striking reciprocity in the patterns of sema6C/6D
expression and the trajectory of proprioceptive axon shafts and collaterals.
Expression of sema6C and sema6D in the developing spinal cord
The observation that proprioceptive axons avoid domains of sema6C/6D expression led us to explore two possible roles for plexinA1 signaling in the patterning of sensory axon projections. We asked first if the emergence of a sema6C/6D-sparse zone in the dorsal spinal cord is required for ingrowth of the collaterals of proprioceptive axons. And we next examined whether sema6C/6D-plexinA1 signaling normally constrains the trajectory of proprioceptive axon shafts, branches, or collaterals within the dorsal spinal cord.
Ectopic sema6C/6D suppresses proprioceptive axon collateral ingrowth
To assess whether the emergence of a sema6C/6D
-sparse zone is necessary for the ingrowth of proprioceptive axon collaterals, we set out to fill this zone by ectopic expression of sema6C/6D throughout the dorsal spinal cord in the chick embryo. Analysis of the chick genome identified a plexinA1
homolog, and a single sema6C/6D
member with a sequence similar to that of mammalian sema6D
. Expression of chick plexinA1
in e8 to e11 DRG was restricted to neurons located in the ventrolateral region (; data not shown), the position of proprioceptive sensory neurons (revealed by runx3
expression; ; Chen et al., 2006
). There was also a complementary relationship between the path of proprioceptive axons (defined by TrkC expression) and domains of high-level sema6D
expression (). Thus, the inverse spatial relationship between sema6C/6D
and proprioceptive axons is conserved in chick spinal cord.
We expressed sema6Cecto
throughout the dorsal spinal cord of chick embryos at e4, prior to the entry of proprioceptive axons (Eide and Glover, 1997
), and assessed domains of sema
expression by co-expression of a GFP
construct. The position of TrkC-labeled proprioceptive, and TrkAlabeled cutaneous, axons was analyzed at e11.0. Ectopic expression of sema6Cecto
resulted in a ~70% suppression in the projection of proprioceptive axon collaterals into the dorsal spinal cord, assessed by quantitation of TrkC immunofluorescence (Figures ). No suppression of TrkC-labeled axon collateral entry was observed after expression of a control GFP
construct, or of sema6Aecto
(Figures ). The actions of sema6Cecto
appeared selective for proprioceptive axons, since their expression did not influence the projection of TrkA+
cutaneous axons into the dorsal spinal cord (Figures S2;). These findings provide evidence that the ingrowth of proprioceptive axon collaterals requires the emergence of a sema6C/6D
-sparse zone in the dorsal spinal cord.
Sema6Cecto and sema6Decto suppress proprioceptive axon collateral projections
Defects in proprioceptive axon projections in plexinA1 mutant mice
We next turned to the issue of whether the trajectory of proprioceptive axons is normally shaped by sema6C/6D-plexinA1 signaling. To assess this, we generated mice targeted with constitutive or conditional plexinA1 mutant alleles (Figures S3A-B). Analysis of the spinal cord of constitutive plexinA1 mutant embryos revealed the absence of plexinA1 immunoreactivity (), establishing that this allele encodes a null mutation. Mice homozygous for the constitutive and conditional plexinA1 alleles were born at Mendelian frequencies, and were viable (data not shown).
In constitutive plexinA1
mutant mice, the specification of proprioceptive and cutaneous neurons occurred normally, as assessed by the presence and number of Pv- and TrkA-labeled DRG neurons, analyzed from e14.5 to p1 (Figures S3C-F; S4A-D; data not shown). Since other plexinA proteins control the peripheral projection pattern of cutaneous sensory neurons (Cheng et al., 2001
; Suto et al., 2005
; Yaron et al., 2005
), we examined whether there is a disruption in the peripheral projections of proprioceptive axons in plexinA1
mutants. To assess this, we crossed a Pv::eGFP
transgene (Dumitriu et al., 2006
) into the constitutive plexinA1
mutant background (Figures S3G-J). Analysis of GFP expression in plexinA1
mutant mice at p1 indicated that the peripherally-directed axons of proprioceptive neurons projected normally, and formed specialized sensory endings with muscle spindles and Golgi Tendon Organs (Figures S3G-J; data not shown). Thus, the early differentiation and peripheral projection of proprioceptive sensory neurons appear unimpaired by the absence of plexinA1 signaling.
Proprioceptive axon collateral trajectory in plexinA1 mutants
We next examined whether the loss of plexinA1 signaling influences the central trajectory of proprioceptive neurons. To define the trajectory of proprioceptive axons before e14.0 we monitored GFP immunoreactivity in mice with a TrkC::eGFP allele crossed into constitutive plexinA1 heterozygote or homozygous mutant backgrounds, and to define axonal projections after e14.5 we monitored expression of Pv.
In plexinA1 mutants examined at e11.5 to e13.0, proprioceptive axons reached the drez (Figures S3K-M; data not shown), segregated from TrkA+ cutaneous axons (Figure S5), and branched rostrocaudally (Figures S3K-M; data not shown), in a pattern similar to that observed in wild-type and plexinA1 heterozygote embryos. And in both plexinA1 heterozygotes and mutants, the collaterals of GFPlabeled proprioceptive axons entered the dorsal spinal cord at e12.5 to e13.5 and projected ventrally (Figures ; data not shown). Thus, plexinA1 signaling is not required for the early positioning of proprioceptive axon collaterals.
Proprioceptive axon projections in the spinal cord of plexinA1 mutant mice
We examined whether the loss of plexinA1 signaling results in later defects in the projections of proprioceptive axon collaterals. A few Pv-labeled axons terminated in the superficial dorsal horn in postnatal plexinA1 mutants (; data not shown), a domain normally devoid of proprioceptive axon terminals (). Nevertheless, analysis of the spinal cord of plexinA1 mutants at p8 revealed that the collaterals of most proprioceptive axons projected to intermediate and ventral target zones in a pattern similar to that observed in wild-type mice (Figures ). Thus, plexinA1 signaling appears not to be involved in establishing the or ventral trajectory of the vast majority of proprioceptive axon collaterals.
Proprioceptive axon shaft positioning in plexinA1 mutants
The loss of plexinA1 signaling did, however, disrupt the organization of proprioceptive axon shafts. In plexinA1 mutants analyzed from e12.5 to e16.5, the shafts of proprioceptive axons were displaced deep into the neuropil of the dorsal spinal cord (Figures ; data not shown). This early disorganization of proprioceptive axonal shafts persisted, and from p0 onwards transversely-orientated shafts of proprioceptive axons were observed to project through much of the superficial dorsal horn on their way to the dorsal columns (Figures ; data not shown). In plexinA1 mutants analyzed at p0 to p8, over 95% of displaced proprioceptive axon shafts were located in the medial half of the superficial dorsal horn (; data not shown), and there was a ~95% decrease in the incidence of proprioceptive axon shafts within the medial half of the dorsal funiculus (Figures ; S6). These findings reveal that the loss of plexinA1 signaling erodes the normal exclusion of proprioceptive axon shafts from the superficial dorsal horn.
is also expressed by cells in the dorsal horn, we were concerned that defects in axon shaft positioning might reflect the loss of protein expression from spinal cord cells rather than sensory neurons. To assess this possibility, we examined proprioceptive axon trajectories in mice in which the plexinA1
gene had been inactivated selectively in DRG neurons. To achieve this, we crossed a conditional plexinA1
mutant line (plexinA1flox
; see Experimental Procedures) with an Ht-PA::Cre
strain that inactivates loxP-flanked genes from DRG neurons, without disrupting expression in spinal cord neurons (Pietri et al., 2003
) (Figures ). At p0, Ht-PA::Cre
mutant mice exhibited a pattern of proprioceptive axon shaft displacement that was similar to that observed in constitutive plexinA1
mutants (Figures ). Thus, the elimination of plexinA1
from proprioceptive sensory neurons is sufficient to induce defects in axon shaft positioning.
A secondary disruption of cutaneous axonal projections in plexinA1 mutants
We next considered the consequences of the displacement of proprioceptive axonal shafts. Since the superficial dorsal horn is the site of termination of many high-threshold cutaneous axons, we examined whether the organization of cutaneous afferent projections is affected in plexinA1 mutants.
We monitored the projection pattern of three classes of cutaneous sensory afferents that project to different laminae within the dorsal horn: unmyelinated sensory axons marked by the binding of isolectin IB4 and the expression of fluoride-resistant acid phosphatase (FRAP) (Nagy and Hunt, 1982
; Molliver et al., 1997
) (Figures ; S8A-H); small-caliber axons marked by expression of substance (SP) and calcitonin-gene-related-peptide (CGRP) (Lawson, 2002
) (Figures ; S9), and thinly myelinated cutaneous axons defined by vGlut1 expression (Todd et al., 2003
) (Figures ; S8I-P). In wild-type and plexinA1
heterozygous mice analyzed at p0, the axons of SP+
sensory neurons have entered the spinal cord, forming diffuse termination zones in the superficial dorsal horn that resolve into more discrete laminar patterns by p8 (Fitzgerald, 1987
; Mirnics and Koerber, 1995
; Lawson, 2002
) (Figures S8A, C, E, G; S9A, C; ; data not shown). Similarly, vGlut1+
sensory axons have entered deeper regions of the dorsal horn by p0, and this pattern sharpens by p8 (Figures S8I, K, M, O; ; data not shown).
Organization of cutaneous afferent projections in plexinA1 mutant mice
In plexinA1 mutants analyzed at p0, the pattern of projection of SP+/CGRP+, IB4+/FRAP+, and vGlut1+ sensory afferents was similar to that in wild-type controls (Figures S8A-D, I-L; data not shown). From p5 onwards, however, we detected a progressive disruption in the pattern of IB4+/FRAP+ and vGlut1+ axonal projections (Figures S8E-H, M-P; ). In plexinA1 mutant mice analyzed at p8, the medial half of the superficial dorsal horn was associated with a zone of exclusion of IB4+/FRAP+ axons (Figures ; data not show). The domain of exclusion of IB4+/FRAP+ axons centered on the location of Pv+ proprioceptive axon shafts, but typically extended beyond the vicinity of the axon shaft itself (Figures ; data not shown). Nevertheless, the lateral domain of the superficial dorsal horn of plexinA1 mutants, which lacks proprioceptive axon shafts, exhibited a normal density of IB4+/FRAP+ axons (Figures ; data not shown). Similarly, there was a local annulus of exclusion of vGlut1+ axons in the medial half of the dorsal horn, centered on the position of proprioceptive axon shafts (Figures ). The spatial link between proprioceptive axon shafts and zones of exclusion of IB4+/FRAP+ and vGlut1+ sensory axons (Figures ) suggests that the disruption of cutaneous axonal projections is a consequence of the displacement of proprioceptive axon shafts, rather than a disruption in the patterning of dorsal horn neurons. Thus, between p5 and p8, two distinct classes of cutaneous afferents that project to different domains of the dorsal horn are excluded from the vicinity of misplaced proprioceptive axon shafts.
In contrast, we did not observe exclusion of SP+/CGRP+ cutaneous afferents from the medial half of the superficial dorsal horn in plexinA1 mutants, assayed at p8 (Figures ; S9; data not shown). Thus, only certain classes of cutaneous sensory axons that project to the superficial dorsal horn appear sensitive to the displacement of proprioceptive axon shafts.
Oligodendrocytes invade the superficial dorsal horn in plexinA1 mutants
How does the displacement of proprioceptive axonal shafts lead to the disorganization of cutaneous projections? One possible scenario is that the presence of proprioceptive axons results in a local disruption in the cellular organization of the dorsal horn itself, which in turn disturbs the projection of cutaneous axons. Against this idea, we found that the density and distribution of dorsal horn neurons at p8, assessed by the pattern of NeuN+ neuronal nuclei, was similar in wild-type and plexinA1 mutant mice (Figures S10A-F). Moreover, the distribution and laminar organization of specific classes of dorsal horn neurons, defined by expression of calretinin, protein kinase CβII and protein kinase Cγ(Ren et al., 1993; Malmberg et al., 1997) was essentially unchanged in plexinA1 mutants (Figures S10G-X), even in domains close to displaced proprioceptive axon shafts (Figures S10G-X).
The preservation of neuronal organization in the dorsal horn of plexinA1
mutants does not exclude an alteration in the patterning of glial cells. During normal development, oligodendrocytes ensheath and begin to myelinate proprioceptive axons during the first few post-natal days (Schwab and Schnell, 1989
; Woodruff and Franklin, 1998
). In contrast, many high-threshold cutaneous axons that project to the superficial dorsal horn remain unmyelinated, and others begin the process of myelination only at a later stage (Schwab and Schnell, 1989
; Woodruff and Franklin, 1998
). Since oligodendrocytes are known to express factors that inhibit the axons of DRG neurons in vivo
and in vitro
(He and Koprivica, 2004
), we considered the possibility that proprioceptive axons might disrupt cutaneous afferent projections through an intermediary action of oligodendrocytes.
To assess this, we monitored the temporal and spatial pattern of oligodendrocyte differentiation in the dorsal spinal cord of wild-type, plexinA1 heterozygote, and mutant mice, assessed by expression of two definitive oligodendrocyte markers-myelin associated glycoprotein (MAG) and myelin basic protein (MBP) (Mikoshiba et al., 1991). In wild-type mice, very few MAG+ and MBP+ oligodendrocytes were detected in the dorsal spinal cord prior to p2, and these few were confined to the dorsal funiculus and dorsal columns (Figures ; S11A). From p3 onwards, there was a progressive increase in the number of MAG+ and MBP+ oligodendrocytes present within the dorsal funiculus and dorsal columns (Figures ; S11B-D), but very few were located in the superficial dorsal horn (Figures ; S11B-D). In contrast, in plexinA1 mutants analyzed from p4 onwards, we observed a >10 fold increase in the number of MAG+ and MBP+ oligodendrocytes within the superficial dorsal horn, compared with heterozygote or wild-type controls (Figures ; S11D-E; data not shown). Over 95% of these ectopic MAG+ and MBP+ oligodendrocytes were found in the medial half of the superficial dorsal horn (Figures ; ). In addition, we found that MAG+ and MBP+ oligodendrocyte processes were tightly associated with displaced Pv+ proprioceptive axonal shafts in the medial half of the superficial dorsal horn (Figures ; ; ; data not shown).
Oligodendrocytes invade the superficial dorsal horn in plexinA1 mutantmice
Foci of ectopic oligodendrocytes coincide with regions of cutaneous afferent disorganization in the dorsal horn of plexinA1 mutant mice
The spatial relationship between foci of ectopic oligodendrocytes and the positioning of IB4+/FRAP+ and vGlut1+ sensory axons differed, however. We found that the presence of MAG+ oligodendrocytes were associated with a broad zone of exclusion of IB4+/FRAP+ axons that often covered most of the medial half of the superficial dorsal horn (Figures ; data not show). In contrast, the zone of exclusion of vGlut1+ axons was occupied almost entirely by MAG+ oligodendrocytes, together with the cell bodies of dorsal horn neurons, assessed by NeuN expression (Figures ). Thus, there is a much more local displacement of vGlut1+ axons.
Finally, we examined whether the ectopic positioning of oligodendrocytes in plexinA1 mutants is a consequence of the loss of plexinA1 expression from oligodendrocytes themselves, or is an indirect response to the displacement of proprioceptive axonal shafts. In wild-type mice, MBP+ oligodendrocytes did not express plexinA1 over the first post-natal week (Figure S12), arguing against the idea that oligodendrocytes respond directly to sema6C/6D signals. Moreover, we detected misplaced MAG+ oligodendrocytes in the vicinity of proprioceptive axonal shafts in the superficial dorsal horn of Ht-PA::Cre; plexinA1flox/flox mice (Figure S13), a situation in which the loss of plexinA1 is restricted to DRG neurons. Thus, the mispositioning of oligodendrocytes appears to be a secondary consequence of the displacement of proprioceptive axon shafts.