appears to guide axons of the nucleus of the medial longitudinal fasciculus (nucMLF) by repulsion and modulation of fasciculation. In
contrast, Sema3D appears to be attractive to telencephalic neurons that form the anterior commissure (AC). Knock-down of
Neuropilin-1A (Npn-1A) phenocopied the effects of Sema3D knock-down on the nucMLF axons, and knock-down of either Npn-1A or
tions among Sema3D, Npn-1A, and Npn-2B. Together, these data support the hypothesis that Sema3D may act as a repellent through
axons interpret multiple molecular signals provided by sur-
rounding cells to navigate to their targets. Semaphorins are one
family of molecules that can direct growing axons. Individual
class 3-secreted semaphorins (Sema3) can function as chemore-
pellants or chemoattractants and sometimes both, depending on
the particular axon type examined (Fiore and Puschel, 2003; Fu-
sists of a neuropilin ligand-binding component, neuropilin-1
(Npn-1) or neuropilin-2 (Npn-2), and a plexin signal-
assays indicate that all class 3 semaphorins tested bind Npn-1
with varying affinities (Feiner et al., 1997; He and Tessier-
Sema3B, -3C, and -3F have been shown to bind Npn-2, whereas
Sema3A does not (Chen et al., 1997; Xu et al., 1998); however,
semaphorins with similar Npn binding affinities in vitro bind to
different cellular sites in situ (Feiner et al., 1997). Moreover,
Npn-2, act as antagonists at Npn-1 receptors and agonists at
studies suggest that binding capability in vitro does not necessar-
ily reveal ligand-receptor function in vivo.
sive activity, based on the similar phenotypes observed in npn-1
al., 1997). Likewise, Npn-2 appears to mediate Sema3F-induced
al., 2003). Explant and growth cone collapse assays suggest that
Sema3C functions through Npn-1–Npn-2 heterodimers (Chen
ual semaphorin acts via different neuropilin complexes to elicit
different axonal responses is unknown.
Here, we demonstrate that Sema3D is required for correct for-
mation of the early axon pathways in the zebrafish brain and that
different functions of Sema3D may require different neuropilin
nucleus of the medial longitudinal fasciculus (nucMLF) and is re-
ciculation of these axons also requires Npn-1A. Results from
Sema3D knock-down and ectopic misexpression experiments sug-
the anterior commissure (AC). Formation of the AC also requires
both Npn-1A and Npn-2B. These results, together with combined
partial knock-down experiments, provide evidence that axons may
respond differentially to a single semaphorin, depending on their
Animals. Zebrafish (Danio rerio) were maintained in a laboratory breed-
ing colony on a 14/10 hr light/dark cycle. Collected embryos were main-
This work was supported by National Science Foundation (NSF) Grant IBN-0110654 and National Institute of
8428 • TheJournalofNeuroscience,September29,2004 • 24(39):8428–8435
Embryo stage was defined as hours post-fertilization (hpf).
In situ hybridization and immunohistochemistry. Digoxygenin-UTP-
labeled riboprobes for sema3D, npn-1A, npn-1B, npn-2A, and npn-2B
were synthesized by in vitro transcription and hydrolyzed to an average
length of ?300 bases by limited alkaline hydrolysis (Cox et al., 1984).
Whole-mount in situ hybridization was performed as described previ-
ously (Halloran et al., 1999).
For whole-mount immunohistochemistry, embryos were fixed in 4%
paraformaldehyde overnight, blocked in 5% sheep serum and 2 mg/ml
BSA in PBS, and incubated overnight (4°C) in the monoclonal antibody
ZN-12 (1:500; Zebrafish International Resource Center, Eugene, OR) or
anti-acetylated ?-tubulin (1:1500; Sigma, St. Louis, MO). Antibody la-
beling was performed with the Vectastain Mouse IgG ABC immunoper-
oxidase labeling kit (Vector Laboratories, Burlingame, CA). For fluores-
Embryos were then incubated simultaneously overnight (4°C) in anti-
acetylated ?-tubulin (1:1500; Sigma) and rabbit-anti-green fluorescent
secondary antibodies (4 ?g/ml; Molecular Probes, Eugene, OR) were
used to bind anti-tubulin and anti-GFP, respectively.
Mosaic Sema3D misexpression. We used the zebrafish hsp70 promoter
(Halloran et al., 2000) to drive ectopic expression of sema3D. Embryos
were injected into the blastomere at the one-cell stage with ?1 nl of a 50
hsp70 promoter and gene expression, embryos were placed in a 39°C
water bath for 1 hr.
Morpholino antisense. Morpholino oligonucleotides against sema3D,
OR). The sequence of the sema3D blocking morpholino (3DMO) is as
follows (with the sequence complementary to the start codon under-
lined): 5?-CATGATGGACGAGGAGATTTCTGCA-3?. The control
morpholino (CONMO) consisted of the identical sequence with four
mispaired bases (lowercase): 5?-CATcATGcACGAGGAGATaTCTcCA-
not overlapping with start codon): 1AMO, (?5) 5?-GAATCC-
TGGAGTTCGGAGTGCG-GAA-3? (?30); 2AMO, 5?-CTTGGTGT-
GATATCCAGAAATCCAT-3?; 2BMO, (?28) 5?-CGCGTAGAGGAA-
AAAGCTGAAGTTC-3? (?52). For the neuropilin morpholinos, a
random sequence was used for a control (STDCON): 5?-CCTCTTAC-
CTCAGTTACA-ATTTATA–3?. To test for morpholino specificity, we
performed BLAST (basic local alignment search tool) analysis of the
morpholino sequences against the Sanger Institute’s zebrafish genome
sequence assembly. None of the morpholinos are predicted to bind to
gene sequences other than those targeted.
Morpholino oligos were injected into newly fertilized embryos at the
1–4 cell stage as described previously (Nasevicius and Ekker, 2000). The
full knock-down: 3DMO (100–500 ?M), 1AMO (250 ?M), and 2BMO
experiment. Subthreshold concentrations used in combination to pro-
duce MLF defects were 25 ?M 3DMO plus 50 ?M 1AMO. The control
?M 1AMO plus 25 ?M STDCON (abbreviated in Fig. 6 as SUBCON).
Concentrations used to cause AC errors were 50 ?M 3DMO plus 100 ?M
1AMO or 250 ?M 2BMO. In all cases, control single subthreshold doses
pholino combined with the appropriate dose of STDCON.
In vitro transcription–translation. Coupled in vitro transcription–
translation (TnT Coupled Reticulocyte Lysate Systems, Promega, Madi-
son, WI) was performed on npn-1A, npn-2A, and npn-2B DNA inserted
tion of biotinylated lysine residues (Transcend tRNA, Promega) during
translation allowed for detection of protein products. Translation was
presence of 10 ?M STDCON or 10 ?M of the appropriate translation-
blocking morpholino. Protein products were separated by SDS-PAGE
cules, CA). The membrane was incubated in streptavidin–HRP (1:5000;
Promega), and protein products were detected via chemiluminescence.
Imaging. All bright-field images were captured on a Nikon TE300
inverted microscope equipped with a Spot RT camera (Diagnostic In-
struments, Sterling Heights, MI) and processed with MetaMorph soft-
ware (Universal Imaging, West Chester, PA). Fluorescent images are
the Bio-Rad 1024 Lasersharp Confocal. Step sizes were 1 ?m.
We previously reported the spatial–temporal expression pattern
of sema3D in the developing zebrafish brain (Halloran et al.,
pathways suggests that it may play a role in their guidance. Spe-
cifically, sema3D is expressed in the neuroepithelium of the ros-
tral ventral midbrain, extending into the ventral diencephalon
(Fig. 1A). Immediately caudal to this domain are the bilateral
nuclei of the nucMLF (Fig. 1B). Each nucMLF extends axons
caudally, away from the sema3D-expressing cells, forming the
two MLFs that extend on either side of the ventral midline.
sema3D is also expressed in the midbrain floor plate, medial to
(Halloran et al., 1999). In addition, sema3D is expressed in the
dorsolateral to the telencephalic neurons that extend to form the
AC (Fig. 1C). Thus, Sema3D is in a position to influence the devel-
opment of multiple axon pathways, including those of the nuc-
MLF and the AC.
The initial tactic that we used to determine whether Sema3D
nents within these neurons. Chick Sema3D has been shown to
Sema3D in chick has been described. Therefore, it is not known
whether Sema3D functions specifically through Npn-1. We ex-
amined the expression of the four zebrafish neuropilins in the
the nucMLF (Fig. 1E) and in the telencephalic neuron clusters
that form the AC (Fig. 1F). npn-2B is also expressed in the telen-
cephalic neurons (Fig. 1H), but not in the ventral midbrain.
telencephalic neurons (Fig. 1G), but it is not expressed in the
ventral midbrain. npn-1B was expressed by neither the nucMLF
nor the telencephalic neurons (data not shown). Together, these
expression patterns suggest that the nucMLF and telencephalic
neurons are likely responsive to class 3 semaphorins.
To investigate the role of Sema3D in guiding axon pathways, we
used morpholino antisense to knock down Sema3D protein. We
examined the effects of Sema3D knock-down by labeling axons
with either the ZN-12 antibody, which labels a subset of early
axon pathways (Metcalfe et al., 1990), or anti-tubulin, which
labels all axon pathways at these stages (Chitnis and Kuwada,
1990). We have shown previously that a morpholino oligo de-
signed against the translation start site of sema3D effectively
blocks Sema3D protein expression (Liu et al., 2004). Embryos
Wolmanetal.•Sema3DandNeuropilinsJ.Neurosci.,September29,2004 • 24(39):8428–8435 • 8429
were injected at the 1–4 cell stage with 3DMO or CONMO and
allowed to develop to specific stages of axon outgrowth. The
throughout the earlier stages of development.
Based on the spatial–temporal relationship between sema3D
expression and the nucMLF axon projection, we hypothesized
that Sema3D acts to repel these axons caudally toward the hind-
brain and spinal cord. Sema3D knock-down caused two types of
guidance defects in nucMLF axon outgrowth. First, in 48% (n ?
63) of 3DMO-injected embryos, we found that one to three
nucMLF neurons extended their axons rostrally into the dien-
cephalon rather than caudally (Fig. 2B, Table 1). This type of
error occurred very rarely (3%; n ? 37) in CONMO-injected
embryos (Fig. 2A, Table 1). These misprojected axons grow
through the ventral midbrain region where Sema3D is normally
expressed, suggesting that it acts to prevent nucMLF axons from
extending in the rostral direction.
In addition to aberrant rostral projections, the nucMLF neu-
rons showed another axon guidance defect after Sema3D knock-
down. In 69% (n ? 139) of 3DMO-injected embryos, the axons
that projected caudally to form the MLF were defasciculated in
the rostral hindbrain (rhombomeres 1–3) (Fig. 2D, Table 1).
the MLF, causing an increase in width of the overall tract. The
defasciculated axons were not biased medial or lateral to the fas-
cicle; rather they were centered on the normal location of the
fascicle. In most CONMO-injected embryos, the MLF axons
form a tight fascicle (Fig. 2C, Table 1); however, we observed a
low degree of defasciculation in 8% (n ? 145) of these controls.
ulation of the MLF in the rostral hindbrain.
Next, we investigated the role of Sema3D in guiding axons of
the telencephalic neurons. At 20–24 hpf, these neurons extend
axons to form the AC, which connects the bilateral telencephalic
neuronal clusters (Chitnis and Kuwada, 1990). Because sema3D
is expressed by the cells dorsolateral to the telencephalic neurons
and near the midline-spanning region of the AC during its for-
mation, we hypothesized that Sema3D knock-down might alter
the development of the AC. In fact, 63% (n ? 39) of 3DMO-
injected embryos exhibited defects in the formation of the AC
(Fig. 2F, Table 1). In these embryos, telencephalic neurons did
not extend axons medially to form the AC, and in some cases the
axons appeared to form swirls and bundles in close proximity to
the clusters of telencephalic neurons (Fig. 2F). Failure of the AC
to develop fully was observed in only 7% (n ? 30) of the control
group (Fig. 2E, Table 1). These results indicate that Sema3D is
required for proper formation of the AC.
views of telencephalon region of 24 hpf embryos. C, In situ hybridization for sema3D shows
form the AC (arrow). E, Cross section of the midbrain shows npn-1A expression (arrow-
(F), npn-2A (G), and npn-2B (H) expression (arrowheads) by the telencephalic neurons.
8430 • J.Neurosci.,September29,2004 • 24(39):8428–8435 Wolmanetal.•Sema3DandNeuropilins
Furthermore, we tested the specificity of these effects of
semaphorin, Sema3A1. sema3A1 is expressed in the region of the
it could potentially affect the development of nucMLF and AC
axons. We injected a morpholino sequence that has been shown
previously to effectively block translation of Sema3A1 (Shoji et
direction of the nucMLF axons, fasciculation of the MLF, or the
observed in Sema3D knock-down are not ubiquitous for all class
3 semaphorins and may be specific to Sema3D loss of function.
consistent with an attractive role for Sema3D in guiding telence-
phalic axons. If Sema3D in the dorsolateral telencephalon acted
to repel axons toward the midline, we might expect to see axons
extending into this dorsolateral domain after Sema3D knock-
down. We never observed dorsolateral axon extension in any of
the 3DMO-injected embryos, suggesting that Sema3D does not
act as a lateral repellant to drive telencephalic axons toward the
midline. Instead, axons remained in close
proximity to their cell bodies, well lateral
to the midline.
To determine whether Sema3D is in-
deed attractive to these axons in vivo, we
saic pattern. We injected a DNA construct
encoding GFP-tagged Sema3D driven by
ran et al., 2000) (hsp:sema3D-gfp) into
1-cell stage embryos. We induced ectopic
Sema3D-GFP expression by heating the
embryos at stages before telencephalic
axon outgrowth and fixed the embryos af-
ter the stage of AC formation. hsp:gfp con-
structs were injected into separate em-
bryos as a control. The embryos were
colabeled with anti-GFP to recognize the
Sema3D-GFP and with anti-tubulin to la-
bel the axon pathways.
In 58% (n ? 19) of the hsp:sema3D-
gfp-injected embryos with cells express-
ing ectopic Sema3D-GFP in the vicinity
of the AC, axons extending from the tel-
encephalic neurons strayed away from
the tract of the AC and grew toward the
ectopic Sema3D-GFP (Fig. 3B,C). On
average, two to five axons deviated from the commissure and
extended toward ectopic Sema3D-expressing cells near the AC
(Fig. 3B,C). One extreme case was observed in which an ec-
topic Sema3D cell located lateral to the telencephalic neurons
caused axons to grow away from the commissure, possibly
preventing commissure formation (Fig. 3C). All of the control
embryos with GFP-expressing cells near the AC (n ? 16) pos-
sessed an established AC in which the axons did not exit the
tract and extend toward the GFP-expressing cells (Fig. 3A).
These results support the hypothesis that Sema3D in the mid-
line region attracts the axons extending from the telencephalic
neurons to form the AC.
development of nucMLF and AC axon pathways in vivo
To gain insight into the specific receptor components that mediate
knock down each of the zebrafish neuropilins expressed in the
nucMLF or telencephalon. As a control we used a random non-
specific 25-mer oligo, STDCON. We tested the efficacy of our
that the morpholinos designed to target npn-1A, npn-2A, and
npn-2B effectively blocked translation (Fig. 4), whereas the
STDCON did not block or reduce translation. Embryos in-
jected with the neuropilin-blocking morpholinos did not show
Knock-down of a critical receptor component for Sema3D
that injection of morpholinos against Npn-1A (1AMO) caused
the same defects in nucMLF and telencephalic axon pathfinding
injected embryos, we found axons extending rostrally from the
nucMLF (Fig. 5B, Table 1). None of the STDCON-injected em-
bryos (n ? 20) displayed this phenotype (Fig. 5A, Table 1). Fur-
thermore, 82% (n ? 112) of 1AMO-injected embryos exhibited
in the presence of a control morpholino or in the presence of morpholinos designed to block
translation of Npn-1A, Npn-2A, or Npn-2B. A 10 kDa band of an endogenously biotinylated
Morpholinos block translation of Npn-1A, -2A, and -2B in vitro. Protein bands
Wolmanetal.•Sema3DandNeuropilinsJ.Neurosci.,September29,2004 • 24(39):8428–8435 • 8431
marked defasciculation of the MLF in rhombomeres 1–3 (Fig.
5D, Table 1) as compared with 0% of controls (n ? 23) (Fig. 5C,
Table 1). Not only did the incidence of MLF defasciculation in-
crease after Npn-1A knock-down as compared with Sema3D
knock-down, but MLF axons in 1AMO-injected embryos were
qualitatively more dramatically defasciculated, spreading over a
Npn-1A knock-down also prevented the formation of the AC in
74% of embryos (n ? 31) (Fig. 5F, Table 1), whereas 10% (n ?
Table 1). These results suggest that like Sema3D, Npn-1A is also
required for proper guidance of nucMLF and AC axons.
Interestingly, we also observed failure of AC formation after
knock-down of Npn-2B. The AC failed to form in 50% (n ? 28)
(Fig. 5G, Table 1) versus 10% (n ? 20) of STDCON-injected
from that of Sema3D or Npn-1A knock-down in which the tel-
encephalic axons formed bundles that did not cross the midline.
Despite npn-2A expression in a subset of telencephalic neurons,
knock-down of Npn-2A did not disrupt AC formation (data not
shown). Thus, Npn-2B, in addition to Sema3D and Npn-1A,
appears to be required for axon growth through the AC.
To further investigate which neuropilins might compose the
Sema3D receptor, we investigated potential interactions among
Sema3D, Npn-1A, and Npn-2B. We performed coinjection ex-
periments with subthreshold doses of morpholinos, in which we
tial knock-down of the neuropilin would enhance the very weak
phenotype caused by partial knock-down of Sema3D. This ap-
proach was designed to mimic double heterozygote studies in
which a genetic interaction would suggest a functional ligand-
receptor pair. As controls, we injected single subthreshold doses
of each morpholino with enough standard control morpholino
jections (SUBCON; see Materials and Methods for concentra-
tions). These subthreshold doses of each morpholino alone (re-
nations of Sema3D, Npn-1A, and Npn-2B caused a strong in-
crease in phenotypic effects.
Combined partial knock-down of Sema3D and Npn-1A
caused defasciculation of the MLF and failure of the AC to de-
velop. In 47% (n ? 34) of SUB3D–SUB1A-coinjected embryos,
we observed a high degree of defasciculation (Fig. 6B, Table 2)
compared with the tightly formed fascicles observed in all em-
bryos injected with a single subthreshold morpholino (n ? 43)
of Sema3D and Npn-1A blocked formation of the AC in 55%
hpf embryos injected with STDCON (A) or 1AMO (B). NucMLF axons extend rostrally after
(C) or 1AMO (D). The MLF is highly defasciculated after Npn-1A knock-down (arrows). E–G,
labeling of 24 hpf embryos injected with SUBCON (A) or SUB3D–SUB1A (B). This SUBCON
embryo was injected with a subthreshold dose of 3DMO mixed with STDCON MO. Embryos
beling of 27 hpf embryos injected with SUBCON (C), SUB3D–SUB1A (D), SUB3D–SUB2B (E),
8432 • J.Neurosci.,September29,2004 • 24(39):8428–8435Wolmanetal.•Sema3DandNeuropilins
(n ? 22) of the injected embryos (Fig. 6D, Table 2) compared
with 21% of control embryos injected with SUB3D alone (n ?
14) (Fig. 6C, Table 2) and 23% of those injected with SUB1A
alone (n ? 13) (Fig. 6C, Table 2). SUB3D–SUB1A-coinjected
embryos, however, did not exhibit rostral extension errors by
nucMLF axons, suggesting that the smaller amounts of endoge-
nous Sema3D and Npn-1A remaining after partial knock-down
may be sufficient to properly direct these axons caudally.
Simultaneous partial knock-down of Sema3D and Npn-2B
not form in 46% (n ? 28) of embryos injected with SUB3D–
with SUB3D alone and 20% (n ? 15) of those injected with
whether there was a genetic interaction between Npn-1A and
Npn-2B, which would suggest that both neuropilins worked to-
(n ? 35) of SUB1A–SUB2B-coinjected embryos (Fig. 6F, Table
(Fig. 6C, Table 2). In each simultaneous partial knock-down ex-
periment, the phenotype observed was indistinguishable from
the previously described full knock-down defects of Sema3D,
Npn-1A, and Npn-2B.
In this study, we define three guidance roles for Sema3D in the
formation of early axon tracts in the zebrafish brain. Sema3D
knock-down indicates that Sema3D is necessary for guidance of
nucMLF axons. First, it contributes to the initial decision of nu-
cMLF axons to extend caudally. Sema3D is expressed rostral to
the nucMLF, and Sema3D knock-down causes some nucMLF
axons to extend aberrantly in the rostral direction, where
Sema3D would normally be expressed. This result provides
strong evidence that Sema3D is repulsive to these axons and is
consistent with our previous results showing that Sema3D repels
retinal ganglion cell axons (Liu et al., 2004). Knock-down of
Npn-1A phenocopied Sema3D knock-down, suggesting that
Npn-1A is a required component of the Sema3D receptor. De-
spite this result, simultaneous partial knock-down of Sema3D
which could argue against Sema3D and Npn-1A acting as a
ligand-receptor pair. Knock-down of the other neuropilins did
not affect the extension direction of nucMLF axons, however,
indicating that they are not required and are likely not serving as
Npn-1A, it would follow the precedent set by the most closely
sive actions in vitro and in vivo (He and Tessier-Lavigne, 1997;
Kitsukawa et al., 1997; Kolodkin et al., 1997; Taniguchi et al.,
1997). The apparent lack of interaction between Sema3D and
Npn-1A could indicate that complete suppression of Sema3D
and/or Npn-1A is required to produce misguided nucMLF
Even after full knock-down of Sema3D or Npn-1A, the ma-
jority of nucMLF axons extended caudally. The incomplete pen-
etrance of this phenotype suggests that these axons are also
guided by other cues. Redundancy in guidance signals might be
Perhaps there are other repulsive cues in the region of Sema3D
by multiple guidance signals. Perhaps the best studied case is the
midline crossing decision of spinal commissural axons. These
axons are attracted to the midline by netrin and sonic hedgehog
(Serafini et al., 1996; Charron et al., 2003) and repelled from the
midline by the combined activity of three slit genes (Long et al.,
2004). Single mutations in any of these genes cause only some or
no commissural axons to make midline errors (Serafini et al.,
1996; Plump et al., 2002; Charron et al., 2003; Long et al., 2004).
most nucMLF axons. Although the morpholinos appeared to
small amounts of protein remain after knock-down in vivo.
Sema3D is also required for the fasciculation of nucMLF ax-
ons following their caudal extension. Fasciculation is important
because late developing axons tend to follow fascicles set by ear-
lier axons (Drazba and Lemmon, 1990) and because it contrib-
utes to correct innervation of targets by regulating where axons
diverge from fascicles to invade target tissues (Tang et al., 1992,
1994; Rutishauser and Landmesser, 1996). Again, Npn-1A ap-
pears to be required for this effect of Sema3D, because Npn-1A
knock-down mimics the defasciculation caused by Sema3D
knock-down. Furthermore, we demonstrated a genetic interac-
tion between Sema3D and Npn-1A in that the combined sub-
threshold morpholinos produced strong MLF defasciculation.
Class 3 semaphorins have been shown previously to be re-
quired for fasciculation. Both Sema3A and Npn-1 knock-out
mice exhibit defasciculation of peripheral–cranial nerves (Kit-
sukawa et al., 1997; Taniguchi et al., 1997), and Sema3F and
Npn-2 knock-out mice show defasciculation of several CNS
pathways (Giger et al., 2000; Sahay et al., 2003); however, the
mechanism by which semaphorins cause fasciculation is still not
well defined. Both Sema3A and Sema3F are expressed in the tis-
sues surrounding specific fascicles, suggesting that they mediate
fasciculation by generating a repulsive surround. Sema3D is ex-
pressed in the midbrain floor plate, medial to the two MLFs, and
could provide a generally inhibitory region around MLF axons.
Sema3D knock-down does not cause MLF axons to converge on
a midline repellant. Rather, the defasciculation occurring after
Sema3D or Npn-1A knock-down is centered on the normal lo-
cation of the fascicles. Moreover, ectopic Sema3D-expressing
cells lying lateral to the MLF did not cause these axons to divert
observations). Together, these results suggest that Sema3D may
not be mediating fasciculation through repulsion. Alternatively,
Wolmanetal.•Sema3DandNeuropilinsJ.Neurosci.,September29,2004 • 24(39):8428–8435 • 8433
activity of adhesion molecules that mediate fasciculation. Evi-
dence for the ability of class 3 semaphorins to regulate adhesion
comes from studies showing that Sema3A directly modulates in-
tegrin activity and the adhesion of vascular endothelial cells
(Serini et al., 2003). Neuropilins, in addition to forming part of
the semaphorin receptor complex, can act as cell adhesion mol-
ecules (CAMs) to mediate axon fasciculation (Fujisawa et al.,
1997) and cell aggregation, and aggregation is not caused by a
2000). Moreover, in some cases, the CAM L1 is a necessary com-
ponent for the Sema3A receptor (Castellani et al., 2000, 2002,
2004). These dual functions for CAMs raise the possibility that
there may exist complex cross-talk between semaphorins and
CAMs, and perhaps semaphorin binding to neuropilin may
modulate the adhesive properties of neuropilin itself or corecep-
also may function as an attractive cue to the telencephalic axons
and after full or simultaneous partial knock-down of Sema3D,
Npn-1A, and/or Npn-2B, axons fail to extend toward the mid-
line. This phenotype is strikingly similar to the loss of the AC in
mutant mice lacking the midline attractant netrin that also dis-
play bundling of AC axons lateral to the midline (Serafini et al.,
1996); however, possible models of Sema3D function other than
direct attraction cannot be ruled out. For example, Sema3D
could antagonize binding of another repulsive semaphorin ex-
pressed at the midline, thus allowing AC axons to cross the mid-
to Npn-1 in vitro (Takahashi et al., 1998); however, our result
showing that axons veered off the pathway of the AC toward
of direct attraction. Interestingly, Sema3F and Npn-2 act as a
Sahay et al., 2003). It appears that Sema3F in surrounding neu-
rons acts as a repellant to funnel axons into the AC. In Sema3F
null mice, in conditional mutants lacking Sema3F only in neu-
rons, or in Npn-2 knock-out mice, AC axons are misdirected or
decussate in a highly disorganized manner (Sahay et al., 2003).
On the contrary, in our knock-down phenotype, axons bundled
may interpret both repulsive and attractive cues to guide AC
The fact that both Npn-1A and Npn-2B are required for AC
formation and show genetic interaction in partial knock-down
form a heterodimer in a functional, attractive Sema3D receptor.
repel axons through Npn-1–Npn-2 heterodimers (Chen et al.,
1998; Takahashi et al., 1998). Npn-1 and Npn-2 are capable of
cells in the absence of ligand (Chen et al., 1998). In addition,
affinities in vitro, and functional assays with neuronal explants
suggest that both Npn-1 and Npn-2 may be required to mediate
the repulsive activity of Sema3C (Chen et al., 1997; Takahashi et
al., 1998). Our in vivo functional data provide evidence to sup-
port the idea that Sema3D-induced attraction is mediated by
Npn-1A–Npn-2B heterodimers. Interestingly, both Sema3B and
Sema3C, like Sema3D, have attractive as well as inhibitory activ-
ities: Sema3B for olfactory neurons (de Castro et al., 1999) and
Sema3C for cortical neurons (Bagnard et al., 1998). It is not
known which receptor components mediate attraction by
Sema3B or Sema3C.
Our results show that a single semaphorin, Sema3D, has the
potential to repel, attract, and modulate adhesion among axons
in vivo. Moreover, our data support a model whereby Sema3D
repels and regulates fasciculation via Npn-1A-containing recep-
tors and attracts via Npn-1A–Npn-2B heterodimers. This model
is reminiscent of netrin guidance, in which axons expressing
DCC (deleted in colon cancer) are attracted to netrin; however,
repelled (Hong et al., 1999); however, no such mechanism has
been demonstrated previously for semaphorins. For a complete
understanding of semaphorin function, it will be crucial to also
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