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First published online 11 June 2008
doi: 10.1242/dev.009019
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Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Author for correspondence (e-mail:
a.j.furley{at}sheffield.ac.uk)
Accepted 15 May 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Axon guidance, Endocytosis, Neural cell-adhesion molecule, Semaphorin, Sensory neurons, Mouse, Cntn2
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Although these afferents arise from initially similar
projections into the dorsal root entry zone (DREZ) followed by bifurcation and
extension in the dorsal funiculus (DF), the subsequent branching of
proprioceptive collaterals into the dorsal horn occurs earlier and more
medially than TrkA+ collaterals (Mirnics
and Koerber, 1995; Ozaki and
Snider, 1997).
Differential responsiveness to spinal cord-derived chemorepellants, notably
semaphorin 3A (Sema3A), may underlie the difference in the pathways taken.
When proprioceptive collaterals are extending into the ventral spinal cord
(Ozaki and Snider, 1997),
Sema3A is expressed in the ventral horn and TrkA+ sensory collaterals remain
restricted to the DF. Correspondingly, both ventral spinal cord and Sema3A
repel NGF-dependent (TrkA+), but not NT3-dependent sensory axons in vitro
(Fitzgerald et al., 1993;
Messersmith et al., 1995;
Pond et al., 2002;
Puschel et al., 1996). Thus,
entry into the dorsal horn was proposed to be determined by programmed
sensitivity to Sema3A (Messersmith et al.,
1995); initially all afferent axons are sensitive to Sema3A, but
NT3-dependent axons lose this sensitivity coincident with collateral
extension, owing to downregulation of a component of the Sema3A receptor,
neuropilin 1 (NRP1) (Fu et al.,
2000; Pond et al.,
2002). Subsequent entry of TrkA+ fibres was suggested to be due to
a general downregulation of Sema3A in the spinal cord
(Messersmith et al.,
1995).
However, although in chick Sema3A is expressed dorsally and then recedes
followed closely by TrkA+ fibre entry (Fu
et al., 2000), in rodents, Sema3A expression is restricted to the
ventral horn from the earliest stages
(Messersmith et al., 1995;
Wright et al., 1995;
Zou et al., 2000). A brief
period of Sema3A expression in cells adjacent the dorsal funiculus (DF) may
explain this in part (Wright et al.,
1995), although mice lacking Sema3A or components of its receptor
(NRP1, plexin A3/A4) have been reported to have relatively minor defects in
the projections of NGF-dependent sensory afferents
(Behar et al., 1996;
Gu et al., 2003;
Kitsukawa et al., 1997;
Yaron et al., 2005). Together,
this suggests that additional repulsive cues exist in the ventral spinal cord
(Anderson et al., 2003;
Masuda et al., 2003) or that
local cues in the dorsal spinal cord play a role in modulating this
process.
One class of molecules expressed on sensory afferents and known to mediate
short-range interactions are the L1-like neural cell adhesion molecules
(L1nCAMs) (Brummendorf and Rathjen,
1996). In chick, injection into the developing spinal cord of
antibodies to axonin 1 or NgCAM (the orthologs of rodent TAG-1 and L1,
respectively) disturbs pathfinding of nociceptive fibres
(Perrin et al., 2001).
However, although it is known that these molecules are binding partners, their
ability to bind homo- and heterophilically
(Brummendorf and Lemmon, 2001;
Brummendorf and Rathjen, 1996),
and their widespread expression in the developing spinal cord and on
innervating sensory axons (Perrin et al.,
2001), makes their precise role and site of action difficult to
determine. The finding that L1 is required for cortical axon responses to
Sema3A (Castellani et al.,
2000) suggests these CAMs may be good candidates for short-range
cues that could modulate responses to diffusible molecules in the spinal cord,
particularly as L1 is known to bind both to NRP1
(Castellani et al., 2000;
Castellani et al., 2002) and to
TAG-1 (Cntn2 - Mouse Genome Informatics)
(De Angelis et al., 2002;
Kunz et al., 1998).
Here, we show that TrkA+ afferents enter the dorsal horn prematurely in both L1 and TAG-1 mutant mice, and that NGF-dependent sensory axons require TAG-1 as well as L1 in order to be repelled by Sema3A. However, we show that, unlike L1, TAG-1 does not bind directly to NRP1 and suggest instead that TAG-1 modulates responses to Sema3A via its interactions with L1. Moreover, in the absence of TAG-1, there is a reduction in the disappearance of L1 and NRP1 from the growth cone surface that normally occurs in response to Sema3A, suggesting that TAG-1 may exert its effect by facilitating the endocytosis of the L1/NRP1 complex. Consistent with this, we find that, after Sema3A treatment, TAG-1 also disappears from the growth cone surface and can be found colocalised inside the cell with clathrin. The effect of loss of TAG-1 on TrkA+ afferent pathfinding is stronger than losing L1 and evidence is presented that TAG-1 is required for responses to other, as yet unidentified repulsive factors in the ventral spinal cord. These findings indicate a wider role for L1nCAMs in modulating responses to diffusible long-range inhibitory molecules.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Poliak et al.,
2003). Mice were maintained on 129SvEv (L1 null; TAG-1 null) or
C57/Bl6 (TAG-1 null; TAG-1a) backgrounds. The phenotypes and data
reported for TAG-1 nulls were the same on C57Bl6 or 129SvEv backgrounds.
TAG-1a mutant allele was generated using a 1.65 kb fragment
5' to EcoRI in exon 3 (13586-13730 in GenBank NC_000067) and
7.5 kb 3' to EcoRI* site in exon 6 (14632-14841)
cloned flanking MC1neopA and flanked by pMC1tk and PGKtk
(Fig. 6). Homologous
recombination in Bruce4 ES cells and germline chimaeras were created as
described (Kontgen et al.,
1993). Routine PCR genotyping was carried out using primers in
exon 5 (5'-GGAGGAGAGAGACCCCGTGAAA-3') and exon 6
(5'-ACACGAAGTGACGCCCATCCGT-3') plus neo-specific primers
(Fig. 6C). Western analysis of
postnatal cerebellum lysates was as described
(Dodd et al., 1988). RT-PCR
(Hybaid protocol) was carried out using postnatal cerebellum RNA using a
primer in exon 2 (5'-TCTCAGTCTCCAGTTGACTCTCCTG-3'), with primers
in exon 8 (5'-GAGTTCTCTGCCTCACATTCATAGG-3'), exon 9
(5'-CATCACAGCCCCAACGTAAGTT-3') or exon 10
(5'-AATCGCAGGTCCCCAGCCAA-3' (a-d respectively in
Fig. 6E). Product from primers
a and d was cloned for sequencing.
Noon of day when copulation plug found designated embryonic day 0.5 (E0.5). Animals generated and maintained with appropriate UK Home Office and Local Ethical Committee approval.
Immunodetection
Antibodies used were TAG-1 [monoclonal (mAb) 4D7, rabbit anti-TAG-1
(Dodd et al., 1988), TG3
polyclonal (Denaxa et al.,
2001)]; L1 [mAb324; Chemicon
(Lindner et al., 1983)];
neurofilament [mAb2H3 (Dodd et al.,
1988)]; TrkA (rabbit anti-TrkA, a gift from L. Reichardt);
neuropilin 1 (rabbit anti-neuropilin 1, a gift from A. Kolodkin); human
immunoglobulin Fc domain (Goat anti-human Fc; Sigma); alkaline phosphatase
(mouse IgM; Sigma). Immunodetection was carried out as described
(Cohen et al., 1998).
DNA constructs, protein production and blocking reagents
DNA expression constructs gifts were as follows: Sema3A from A.
Püschel (Puschel et al.,
1996); full-length and soluble (AP and Fc fusions) neuropilin 1
(NRP1) and NRP2 from A. Kolodkin (Gu et
al., 2003; Kolodkin et al.,
1997); full-length rat TAG-1 in pcDNA-1 from R. Jia and T. Jessell
(Furley et al., 1990);
full-length ratL1 from D. Felsenfeld; huL1-Fc fusion from S. Kenwrick
(De Angelis et al., 1999);
full-length plexin A4 from A. Yaron and M. Tessier-Lavigne
(Yaron et al., 2005); TAG-1-Fc
fusion as described (Poliak et al.,
2003). Cos7 cells were transiently transfected using lipofectamine
(Invitrogen). Sema3A production was achieved using hanging-drop aggregates as
described (Anderson et al.,
2003); the amount of DNA transfected was titrated to produce
repulsion of dorsal root ganglion (DRG) axons comparable with ventral spinal
cord (VSC) explants co-incubated at a similar distance (cf.
Anderson et al., 2003).
Cell-binding assays with soluble ectodomain fusions as described
(Anderson et al., 2003;
Tamagnone et al., 1999).
Growth cone collapse assays used Sema3A/mock-conditioned media from
lipofectamine-transfected HEK293T cells
(Kitsukawa et al., 1997).
TAG-1 function was blocked using monoclonal (4D7) and polyclonal (TG3)
antibodies titrated for each repellant source to give maximal repulsion
blockade. Glycosyl phosphatidylinositol (GPI) anchored membrane proteins were
cleaved using phosphatidylinositol-specific phospholipase C (PI-PLC) from
Bacillus thuringienis (Stoeckli
et al., 1996).
Axon repulsion and growth cone collapse assays
Collagen co-culture axon repulsion assays as described
(Messersmith et al., 1995).
DRG and VSC dissected from thoracic segments of E13.5 embryos. DRG explants
were positioned 200-300 µm from VSC or mock-/Sema3A-transfected Cos7 cells
and cultured with 50 ng/ml nerve growth factor (NGF) for 36 hours. Axons were
counted in proximal (P; Fig.
3D) and distal (D) quadrants, then P/D ratio calculated. Data were
analysed using Student's paired t-test (P versus D axons), Student's
unpaired t-test (to compare mean P/D ratios between DRG from
different genotypes) and ANOVA (to standardise separation distances). Similar
results obtained when axon lengths were measured (not shown).
Growth cone collapse: E13.5 DRG were similarly cultured on
PDL/laminin-coated coverslips. After 
18 hours of growth, Sema3A- or
mock-conditioned media were applied for 30 minutes, before fixation with 4%
PFA. The percentage of collapsed growth cones was calculated and differences
analysed using Student's t-test.
Analysis of endocytosis
DRG were cultured as in the collapse assays; however, the Sema3A treatment
lasted for 45 minutes. Cultures were stained on ice using anti-L1 and
anti-NRP1 or anti-TAG-1 in L-15 for 20 minutes, fixed in 4% PFA and the
secondary antibody [including FITC-Phalloidin (SIGMA), where appropriate] was
applied. Lack of neurofilament staining confirmed inaccessibility of internal
proteins. For experiments examining TAG-1/clathrin colocalisation, explants
were treated with Sema3A for 10 minutes, fixed in 4% PFA, permeabilised,
stained overnight in anti-clathrin heavy chain (X22, a gift from E. Smythe)
and anti-TAG-1, then stained with appropriate secondary antibodies. Images
were captured using a Volocity Grid Confocal (Improvision) on an Olympus BX61.
Image analysis was performed using Volocity Classification. Briefly, average
intensity of signal per unit area for the distalmost 50 µm of a growth cone
was normalised to the average intensity of a 300 µm2 region of
cell-free background. P-values were calculated by use of either
one-tailed (wild type versus wild type+Sema) or two-tailed (wild type+Sema
versus null+Sema) Student's t-tests with Welch's correction (to
account for differences in variance) being applied where appropriate. Values
plotted (Fig. 8) are
mean–s.e.m.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), but not in mouse. We compared expression of TAG-1 and L1 on
TrkA+ afferents on embryonic days 12.5 to 15.5 (E12.5-E15.5;
Fig. 1). For simplicity, the
different fibre subsets will be referred to as TrkA+ and NGF dependent (to
include both mechanoreceptive and nociceptive neurons) or TrkA-
(proprioceptive, Ia, TrkC+ or NT3 dependent neurons).
In the thoracic spinal cord, afferents of early-born proprioceptive neurons
arrive in the DREZ just before E10.5 and bifurcate to form the primordial
dorsal funiculus [DF (Ozaki and Snider,
1997)]. New afferents continue to arrive and bifurcate over the
next few days, taking progressively more lateral positions in the DF, the
later-arriving and more lateral being strongly TrkA+
(Fig. 1A,C,D)
(Ozaki and Snider, 1997;
Perrin et al., 2001). By
E13.5, the first arriving TrkA- afferents begin to extend collaterals into the
dorsal horn, reaching motoneuron targets by E15.5 (arrows in
Fig. 1J,L,N,P). By contrast,
the very first TrkA+ collaterals do not begin to appear in the dorsal horn
until E14.5-E15.5 (Fig. 1I,K,L)
and are not abundant until later stages (see below).
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)], by E12.5 its expression appears to be graded from high
laterally to low or absent medially (Fig.
1E,G,H; asterisk indicates an L1+ TAG-1-region) and by E15.5 this
difference becomes marked and the most medial fibres express only L1
(asterisks in Fig. 1H-N). These
TrkA-, L1+ fibres (Fig. 1K,L)
are almost certainly proprioceptive axons; their collaterals can be seen
extending ventrally through the dorsal horn towards targets in the ventral
spinal cord (Fig. 1I-P,
arrows). By contrast, all TrkA+ fibres express TAG-1 and L1, including the few
collaterals that have begun to extend (arrowheads in
Fig. 1I,L,M,P). Therefore,
TrkA+ afferents co-express TAG-1 and L1, whereas the TrkA- afferents continue
to express L1, but downregulate TAG-1 at the time of collateral extension into
the dorsal horn.
TrkA+ afferents project prematurely in TAG-null and L1-null mice
To determine whether loss of L1 or TAG-1 has an effect on TrkA+ sensory
afferent guidance, we followed the path of these fibres in null mutant animals
(Cohen et al., 1998;
Poliak et al., 2003)
(Fig. 2). Significant numbers
of TrkA+ axons were found projecting into the dorsal horn from the DF at E12.5
and E13.5 in animals lacking either L1 or TAG-1, whereas few if any such
projections are found in wild-type littermates
(Fig. 2A-H,M). By E14.5,
although a few TrkA+ collaterals were now present in wild-type animals,
considerably more were found in the mutants
(Fig. 2I,K-M). At this latter
age, not only were aberrant projections more prevalent, but they were also
significantly greater in length (Fig.
2N), sometimes projecting almost to the midline on both sides of
the spinal cord in TAG-1 mutants (Fig.
2I,K). Aberrant projections also occurred all along the
mediolateral extent of the TrkA+ region of the DF, with as many as 40% of
projections located medially at E14.5 compared with only 5% in wild type
(Fig. 2O). Even at E12.5 as
many as 20% could be found in the medial half of the dorsal horn (not shown),
suggesting that in mutants these projections are aberrant, rather than the
result of accelerated development.
There was no obvious qualitative difference in the aberrant projections in TAG-1 nulls compared with L1 nulls - in both cases length and projection angle varied considerably - but quantitation of the number of such projections revealed significantly more in the TAG-1 mutants at all ages [as many as 70% more projections in TAG-1 versus L1 nulls (E13.5); t-test, P<0.001; Fig. 2M].
TAG-1 and L1 null sensory axons fail to respond to ventral spinal cord
Our observations indicate that TAG-1 and L1 are required to prevent
premature entry of TrkA+ sensory afferents axons into the dorsal horn, in
broad agreement with antibody perturbation experiments carried out in chick
(Perrin et al., 2001). This
could reflect a requirement for these molecules to mediate interactions with
binding partners in the dorsal horn
(Perrin et al., 2001).
Alternatively, these molecules may be necessary for axonal responses to
diffusible chemorepellants emanating from the ventral spinal cord [VSC
(Messersmith et al., 1995)],
especially as cortical axons from neonatal mice lacking L1 are reported to be
insensitive to Sema3A (Castellani et al.,
2000).
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0.70 for L1-null axons and 1.00 for TAG-1
null axons, indicating, respectively, partial or complete loss of repulsion by
VSC. The difference between these results is significant (P<0.01,
unpaired t-test), suggesting that L1 null axons remain partially
responsive to VSC-derived repellants (Fig.
3E). Similar results were obtained by blocking TAG-1 function
using monoclonal (4D7) or polyclonal (TG3) anti-TAG-1 antibodies or
phosphoinositol-specific phospholipase C (PI-PLC) to cleave TAG-1 from the
cell surface (Fig. 3E; not
shown), though in these cases the axons consistently remained partially
responsive. Thus, acute loss of TAG-1 function, as well as long-term genetic
ablation of the TAG-1 gene, leads to a loss of response to VSC. Therefore,
NGF-dependent sensory axons require both L1 and TAG-1 to be fully responsive
to VSC-derived chemorepellants.
NGF-dependent sensory axons lacking TAG-1 fail to respond to Sema3A and other chemorepellants
The repulsion of NGF-responsive sensory afferents by VSC has been shown to
be mediated in part by Sema3A (Fu et al.,
2000; Messersmith et al.,
1995; Shepherd et al.,
1997). However, the differential loss of response to VSC explants
exhibited by L1 compared with TAG-1 null DRG
(Fig. 3E) suggested the
possibility that other chemorepellants were acting in our assay, in accordance
with other studies (Masuda et al.,
2003) and the fact that this region expresses multiple secreted
inhibitory molecules (Zou et al.,
2000). To test whether the altered responses of sensory neurons
from L1 and TAG-1 mutant mice could be attributed to changes in response to
Sema3A, we assayed their response to Sema3A expressed in Cos7 cell
aggregates.
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),
E13.5 L1-null sensory axons were completely refractory to Sema3A-mediated
repulsion (Fig. 4B,E), even at
levels of Sema3A expression that repel wild-type axons
(Fig. 4A) to the same or to a
greater extent than VSC under the same conditions [P/D ratio of 0.096
(Fig. 4E), compare with 0.269
(for VSC versus wild-type axons; Fig.
4G)]. Indeed, the P/D ratio obtained with L1-null, NGF-dependent
sensory axons was not significantly different from that found when Sema3A
activity was blocked by the addition of soluble NRP1 ectodomain-alkaline
phosphatase fusion protein [NRP1-AP (Chen
et al., 1998)] or when DRG were cultured alone
(Fig. 4B,D,E; not shown). The
response of E13.5 TAG-1 null, NGF-dependent sensory axons was also
significantly reduced, though to a lesser degree than L1-null axons (P/D=0.71;
Fig. 4C,E). To demonstrate that
this is a direct effect on growth cone response to Sema3A, we compared TAG-1
mutant growth cones with wild-type in a growth cone collapse assay. As has
been shown previously for L1-null growth cones
(Castellani et al., 2000), the
proportion of growth cones collapsing in response to Sema3A was significantly
reduced in TAG-1-null mutants (Fig.
4H,I). Thus, NGF-dependent embryonic sensory axons require both
TAG-1 and L1 to be fully responsive to Sema3A.
Because the contribution of Sema3A to VSC repulsion of DRG axons is
reported to vary according to embryonic age
(Masuda et al., 2003), we
established its specific contribution in our assay. The repulsion of wild-type
axons by E13.5 VSC was substantially reduced in the presence of soluble
NRP1-AP (Fig. 4F,G), although
significant repulsion remained even at concentrations of NRP1-AP that
completely blocked the strong repulsion by Sema3A-transfected Cos7 cells noted
above (compare Fig. 4E,G),
suggesting that other repellants are active in our assay. The amount of
repulsion remaining after blockade of Sema3A was similar to that when L1
function was lost (Fig. 4G).
Together with our data and previous data
(Castellani et al., 2000)
showing that L1 is required for responses to Sema3A, this suggests that the
loss of response to VSC seen with embryonic L1-null sensory axons is due to
loss of sensitivity to the Sema3A synthesised in these tissues.
Whereas the loss of response of L1-null axons to VSC was partial, TAG-1
null axons showed no response at all (P/D 1.0), a significantly greater
loss than when L1 was lost or when Sema3A was blocked
(Fig. 4G). This indicates both
that TAG-1 is required for responses to Sema3A expressed in embryonic VSC and
that it is probably required for responses to other VSC-derived
chemorepellants. The identity of these latter factors remains unknown, but
they are unlikely to be other Sema3 class molecules as addition of soluble
NRP2-Fc had no effect on VSC repulsion (not shown).
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TAG-1 does not bind directly to Sema3A, neuropilin 1 or plexin A4
L1 has been shown to bind directly to NRP1 and mediate its Sema3A-induced
endocytosis (Castellani et al.,
2000; Castellani et al.,
2002; Castellani et al.,
2004). To establish whether TAG-1 also binds components of the
Sema3A receptor expressed by sensory axons
(Yaron et al., 2005), we
assayed the binding of soluble protein to full-length proteins expressed on
the surface of Cos7 cells (Fig.
5). As expected, this assay demonstrated the homophilic binding of
both L1 and TAG-1, and heterophilic binding between the proteins
(Fig. 5A,B,D,E). We were also
able to demonstrate binding of soluble L1-Fc to membrane-bound NRP1
(Fig. 5C). By contrast, soluble
TAG-1-Fc did not bind either NRP1- or plexin A4-expressing cells
(Fig. 5F,L), even though the
same protein bound L1- and TAG-1-expressing cells at relatively high frequency
(Fig. 5G,H). Similarly, whereas
flag-tagged Sema3A collapsed growth cones, it did not bind to cells expressing
TAG-1 (Fig. 5J,M). Thus,
although we were able to confirm the binding of L1 to NRP1
(Castellani et al., 2000;
Castellani et al., 2002), our
data indicate that TAG-1 binds neither NRP1, plexin A4 or Sema3A.
Deletion of L1-binding domains from TAG-1 is sufficient to disrupt response to Sema3A
TAG-1 might be involved in responses to Sema3A through its known cis
interactions with L1 (Bizzoca et al.,
2003; Buchstaller et al.,
1996; Malhotra et al.,
1998; Rader et al.,
1996). Binding of TAG-1 to L1 is mediated by the first four Ig
domains of TAG-1 and deletion of any of these disrupts binding
(De Angelis et al., 1999;
Rader et al., 1996).
Therefore, we made use of a hypomorphic targeted TAG-1 allele which disrupts
these domains. This allele, which we call TAG-1a, was created
through deletion of part of the TAG-1 locus between exons 3 and 6 that encodes
the first three Ig domains (Fig.
6A). Aberrant splicing of exon 2, which encodes the start codon
and leader sequence, to exons 7 or 9 in these mutants
(Fig. 6E-H) results in the
production of truncated anti-TAG-1 immunoreactive proteins, lacking the first
two (minor product) or three Ig domains (major product) but retaining the
leader sequence (Fig. 6H,J) at
50% of the wild-type level (Fig.
6D; data not shown). We confirmed that these truncated protein
products reach the cell surface by staining live cells in conditions in which
intracellular proteins are not detectable
(Fig. 6I). Thus,
TAG-1a homozygous mice (TAG-1a/a) make no detectable
wild-type TAG-1 protein (Fig.
6D), but instead produce truncated proteins lacking the domains
necessary for binding to L1. As reported for the TAG-1-null allele
(Poliak et al., 2003),
TAG-1a homozygotes are viable and without overt phenotype as
adults.
We tested whether deletion of the L1-binding domains of TAG-1 is sufficient to mimic complete loss of TAG-1 protein in responses to Sema3A. In co-cultures with Sema3A-expressing Cos7 cells, NGF-dependent sensory axons from TAG-1a/a embryos displayed a significant decrease in response, comparable with that seen with TAG-1 null axons and likewise significantly less than the loss seen with L1-null axons (Fig. 7A,B). This indicates that loss of the L1-binding domains of TAG-1 indeed has an effect on axonal Sema3A responses equivalent to complete loss of TAG-1 protein.
TAG-1 mediates responses to non-Sema3A repellants via domains other than those that bind L1
Experiments above suggested that Sema3A was only partially responsible for
the repellant activity of VSC and that TAG-1, but not L1, was important for
responses to both the Sema3A and the non-Sema3A components of the repulsion
derived from this tissue. If loss of the L1-binding domains of TAG-1 was
simply equivalent to loss of all TAG-1 protein, TAG-1a/a sensory
axons should also display a total loss of response to VSC. However, if TAG-1
is involved in chemorepellant responses independent of L1 and Sema3A, deletion
of the L1-binding domains may not affect this function. To test this
possibility we assayed responses of TAG-1a/a sensory axons to VSC
and compared these with our previous results. Although TAG-1a/a
axons showed a significant decrease in response, this decrease was
significantly less than that seen with TAG-1 null axons and similar to that
seen with L1-null axons (Fig.
7C,D). Thus, in response to VSC, TAG-1a/a axons behave
comparably with L1-null axons and with wild-type axons when Sema3A activity is
blocked (Fig. 4G), but retain a
residual response that is completely lost in TAG-1-null axons. This is
consistent with TAG-1 being necessary for responses to Sema3A via its
L1-binding domains, but also being required for responses to other, non-Sema3A
chemorepellants through an L1-independent mechanism.
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; Faivre-Sarrailh et al.,
2000; Poliak et al.,
2003), we considered the possibility that TAG-1 might affect
levels of L1 protein expression. However, we were unable to detect any obvious
change in L1 expression in TAG-1 mutants on cryostat sections or by western
blotting (not shown). We were also unable to see any change in the amount of
L1 protein reaching the plasma membrane of cultured TAG-1-null sensory axons
labelled in conditions in which only extracellular proteins were detectable
(Fig. 8A,B), which we confirmed
quantitatively by structured illumination (GRID) confocal microscopy
(Fig. 8I; see Materials and
methods). Thus, loss of TAG-1 does not appear to affect levels of L1 at the
cell surface of normally growing axons.
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; Castellani et al.,
2004), and because Sema3A binding to NRP1 leads to
co-internalisation with L1 in Cos7 cells
(Castellani et al., 2004), we
considered whether loss of TAG-1 might affect this internalisation. We first
established that, as for L1, levels of NRP1 at the cell surface in TAG-1
mutants were similar to those in wild-type growth cones
(Fig. 8C,D,J). We next
determined whether the internalisation of L1 and NRP1 in response to Sema3A
was affected by loss of TAG-1. We showed first that our system of quantitation
could detect the disappearance of L1 and NRP1 from the cell surface; the
relative intensity of both L1 and NRP1 fluorescence dropped significantly
after Sema3A treatment (Fig.
8E,G,I,J), presumably reflecting internalisation. By contrast, no
such decrease occurred when growth cones lacking TAG-1 were treated
(Fig. 8F,H,I,J). Together, this
suggests that TAG-1 modulates L1-mediated responses to Sema3A by facilitating
the internalisation of L1 and NRP1. Consistent with this reflecting a decrease
in the levels of endocytosis normally seen after Sema3A treatment
(Fournier et al., 2000), we
also saw a decrease in the internalisation of FITC-labelled dextran in
Sema3A-treated growth cones from TAG-1-null mice
(Fig. 8K). Similar to L1 and
NRP1, TAG-1 substantially disappears from the cell surface of Sema3A-treated
wild-type growth cones (Fig.
8L) and, moreover, can be found colocalised with clathrin heavy
chain in the cytoplasm of these collapsed structures
(Fig. 8M-Q). Together, these
data suggest that TAG-1 controls responses to Sema3A in NGF-dependent sensory
growth cones by modulating the endocytosis of L1 and NRP1, and is itself
endocytosed in these responses. |
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Hopker et al.,
1999; Shewan et al.,
2002). Previous studies have begun to uncover the intracellular
signalling mechanisms involved in this integration
(Castellani et al., 2002;
Hopker et al., 1999;
Shewan et al., 2002). Here, we
focused on mechanisms that operate at the plasma membrane to modulate guidance
responses. We have shown that responses of NGF-dependent sensory axons to
diffusible chemorepellants present in the spinal cord are dependent on both L1
and TAG-1, and that loss of either leads to premature innervation of the
dorsal horn. We confirmed that a key component of VSC repulsion is Sema3A and
that L1, which associates with the Sema3A receptor NRP1
(Castellani et al., 2000), is
required for sensory axon repulsion by Sema3A. We showed for the first time
that TAG-1 is also required for these responses but that it does not to bind
directly to Sema3A, NRP1 or plexin A4. Instead, our evidence suggests that the
known cis interaction of TAG-1 with L1
(Buchstaller et al., 1996;
De Angelis et al., 2002)
regulates the co-internalisation of L1 with NRP1 from the cell surface. Our
evidence also suggests that TAG-1 may be involved in responses to other, as
yet unidentified, chemorepellants.
TAG-1 and L1 are required for timely dorsal horn innervation
Experiments using antibodies to axonin 1 (TAG-1) or NgCAM (L1) on chick
embryonic spinal cord in ovo (Perrin et
al., 2001) or in vitro (Shiga
et al., 1997) indicated a role for these molecules in TrkA+
sensory axon pathfinding. Our findings are consistent with these studies in
indicating that these L1nCAMs are required to prevent the premature entry of
TrkA+ axons into the dorsal horn; injection of axonin 1 antibodies into chick
embryos resulted in premature projections comparable in number with those
observed in TAG-1 null mice [40 premature projections per 600 µm of spinal
cord at E6.5 in chick (Perrin et al.,
2001) compared with at least 90 per 600 µm at E14.5 in mice]
and focused around points of dorsal root entry. However, whereas these studies
suggested that TAG-1 and L1 are involved in providing `positive' guidance cues
in order to retain axons in the dorsal funiculus, most probably through
selective fasciculation (Perrin et al.,
2001; Shiga et al.,
1997), our in vitro explant culture evidence indicates that these
molecules are required on sensory axons for responses to diffusible `negative'
cues present in the spinal cord; NGF-dependent axons lacking L1 or TAG-1 are
partially or completely unresponsive to ventral spinal cord-derived
chemorepellants, respectively.
At least part of this loss of response is due to loss of response to Sema3A
that, consistent with previous studies
(Messersmith et al., 1995;
Puschel et al., 1996), we find
to account for a substantial proportion of the VSC repulsive activity at this
stage in rodents. For axons lacking L1, the proportion of response to VSC lost
correlates exactly with the proportion due to Sema3A repulsion, as judged by
blockade with soluble NRP1 ectodomain. Such axons also no longer respond to
soluble Sema3A. This agrees with previous studies demonstrating a requirement
for L1 in responses to Sema3A in postnatal cortical and sensory axons,
mediated by direct binding of L1 to NRP1
(Castellani et al., 2000;
Castellani et al., 2002).
In the case of NGF-dependent sensory axons lacking TAG-1, the loss of response to VSC repulsion is complete and significantly greater than that seen with axons lacking L1. Correspondingly, the loss of response cannot be accounted for simply by loss of response to Sema3A; as indicated above, soluble NRP1 was insufficient to block VSC-derived repulsion completely, even at concentrations of NRP1 that completely block the stronger repulsion seen with Sema3A-transfected Cos7 cells. Moreover, although axons lacking TAG-1 are less responsive to Sema3A-transfected Cos7 cells, the loss of response is less than that seen with L1 mutant axons under the same assay conditions, the reverse of the situation seen when VSC is the source of repellant. This indicates that Sema3A, or indeed other NRP1-binding semaphorins cannot account for all VSC repulsion at this age, and that TAG-1 is required for responses to an unidentified repulsive activity as well as to VSC-derived Sema3A. The greater loss of response to VSC repulsion in TAG-1 nulls compared with L1 nulls is consistent with the greater number of TrkA+ axons prematurely entering the dorsal horn in TAG-1 nulls.
|
). In this
study, as with ours, antibodies to axonin-1/SC2 diminished responses to VSC
repulsion. However, in contrast to our results, these authors argue against a
role for TAG-1 in mediating responses to Sema3A, in part because antibodies to
axonin 1/SC2 did not significantly block responses to Sema3A-transfected Cos7
cells, and because responses of DRG to VSC were not affected by genetic loss
of NRP1 or Sema3A. Two key differences between the studies may account for the
apparent discrepancy. First, our study tested E13.5 VSC with DRG of the same
age and species, whereas the earlier study mixed both ages and species. This
is important because there are significant differences in the expression
profiles of Sema3A and NRP1 both between the species [compare Zou et al. with
Fu et al., for example (Fu et al.,
2000; Zou et al.,
2000)] and at different ages in the same species
(Pond et al., 2002;
Puschel et al., 1995;
Puschel et al., 1996;
Wright et al., 1995;
Zou et al., 2000). In
particular, Masuda et al. make the argument that because E5 chick DRG are
still repelled by E11.5 VSC from Sema3A-null mice, Sema3A has no role in VSC
repulsion at this time and that therefore, because axonin 1/SC2 antibodies do
block repulsion by E11.5 VSC, axonin 1/SC2 may have no role in Sema3A
responses. However, Sema3A is not expressed at high levels in mouse VSC until
E12.5 or later (Puschel et al.,
1995; Zou et al.,
2000); therefore, it is not possible to draw conclusions about the
role of TAG-1 in responses to Sema3A from these data. Second, in our study we
focussed on NGF-dependent (TrkA+) sensory axons known to respond to Sema3A
(Messersmith et al., 1995),
whereas Masuda et al. included both NGF and NT3 in their cultures, and it is
known that NT3-dependent axons lose their response to Sema3A with age
(Pond et al., 2002).
TAG-1 affects responses to Sema3A by modulating the internalisation of the L1/NRP1 complex
Disruption of TAG-1 function by gene knockout is not as effective in
blocking the Sema3A response as is genetic removal of L1 function. This
indicates that, although TAG-1 is required for the full Sema3A response, some
residual activity remains in its absence. Together with our observation that
TAG-1 does not appear to bind directly to Sema3A, NRP1 or plexin A4, this
suggests that TAG-1 influences responses to Sema3A indirectly via its ability
to bind L1 on the same membrane (i.e. in cis)
(Brummendorf and Rathjen, 1996;
De Angelis et al., 1999).
Consistent with this, disruption of the domains known to be important for L1
binding (De Angelis et al.,
1999; Rader et al.,
1996) in our TAG-1a mutant reduces responses to Sema3A
as effectively as complete loss of TAG-1.
How might TAG-1 binding to L1 affect Sema3A responses? One possibility is
that TAG-1 is required for the trafficking of L1 to the cell surface, by
analogy with the role of F3/contactin in the trafficking of CASPR/paranodin
(Faivre-Sarrailh et al.,
2000). However, we could find no significant change in the levels
of cell surface L1 in TAG-1 mutants. However, it has been reported that L1 is
co-internalised with NRP1 in response to Sema3A
(Castellani et al., 2004) and
that Sema3A treatment induces endocytosis locally
(Fournier et al., 2000).
Consistent with this, we found that cell surface levels of both L1 and NRP1
were significantly reduced after Sema3A treatment of wild-type sensory growth
cones, but this reduction did not occur in growth cones lacking TAG-1.
Moreover, the proportion of growth cones taking up FITC-dextran after Sema3A
treatment was reduced in the absence of TAG-1. This suggests that the binding
of TAG-1 to L1 on the neuronal surface may facilitate endocytosis of the
Sema3A receptor complex. Whether TAG-1 directly associates with the
L1-NRP1-plexin A4 complex or modulates the participation of L1 in the complex
indirectly is unclear, although our data do indicate that TAG-1 itself is
endocytosed and becomes associated with clathrin after Sema3A treatment.
Interestingly, because of its known association with lipid rafts
(Kasahara et al., 2000), one
possibility is that TAG-1, through its binding to L1, may recruit components
of the Sema3A receptor into these membrane microdomains, which have been
suggested to be portals for endocytosis
(Parton and Richards, 2003)
and which are known to be required for Sema3A-mediated growth cone repulsion
(Guirland et al., 2004).
Further analysis will be necessary to determine the exact mechanism by which
TAG-1 exerts its effect.
The role of L1nCAMs in axon pathfinding in vivo
Despite our evidence that TAG-1 and L1 expression is required to prevent
premature entry of TrkA+ sensory afferents into the dorsal horn, their exact
role in controlling the pathfinding of these axons remains unclear. Of
particular interest is why molecules known to mediate fasciculation modulate
responses to soluble chemorepellants. One possibility is that this may render
axonal responses context dependent, as suggested by the inhibition of growth
cone collapse by Sema3A by soluble L1
(Castellani et al., 2000;
Castellani et al., 2002). Thus,
TrkA+ growth cones fasciculating on L1-expressing proprioceptive axons as they
enter and bifurcate in the DREZ may be insensitive to local Sema3A
(Fu et al., 2000;
Wright et al., 1995), so long
as they maintain their TAG-1/L1-mediated fasciculation. Accordingly, the
aberrant TrkA+ projections seen in TAG-1 and L1 mutant mice were most evident
at the entry point (Fig. 2)
(Perrin et al., 2001),
suggesting that these axons fail to bifurcate. Subsequent downregulation of
Sema3A (Wright et al., 1995)
normally would release this restraint and allow collateral formation
(Messersmith et al., 1995).
Together with recent evidence from zebrafish that class 3 semaphorins may
regulate L1 expression (Wolman et al.,
2007), this suggests that the mechanisms by which semaphorins and
adhesion molecules regulate fasciculation are more intricate than a simple
balance of repulsion and adhesion (Yu et
al., 2000).
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
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